Methods for attenuating release of inflammatory mediators and peptides useful therein

ABSTRACT

The present invention includes methods of inhibiting or suppressing cellular secretory processes. More specifically the present invention relates to inhibiting or reducing the release of inflammatory mediators from inflammatory cells by inhibiting the mechanism associated with the release of inflammatory mediators from granules in inflammatory cells. In this regard, the present invention discloses an intracellular signaling mechanism that illustrates several novel intracellular targets for pharmacological intervention in disorders involving secretion of inflammatory mediators from vesicles in inflammatory cells. Peptide fragments and variants thereof of MANS peptide as disclosed in the present invention are useful in such methods.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. Ser. No. 60/833,239filed on Jul. 26, 2006, which is incorporated in its entirety byreference.

FIELD OF INVENTION

The present invention relates to peptides or peptide compositions andmethods of their use to attenuate (or inhibit or reduce) the stimulatedrelease of mediators of inflammation from inflammatory cells duringinflammation. The present invention also relates to use of thesepeptides or peptide compositions to modulate an intracellular signalingmechanism regulating the secretion of inflammatory mediators frominflammatory cells.

BACKGROUND OF THE INVENTION

Inflammatory leukocytes synthesize a number of inflammatory mediatorsthat are isolated intracellularly and stored in cytoplasmicmembrane-bound granules. Examples of such mediators include, but are notlimited to, myeloperoxidase [MPO] in neutrophils (see, for example,Borregaard N, Cowland J B. Granules of the human neutrophilicpolymorphonuclear leukocyte. Blood 1997; 89:3503-3521), eosinophilperoxidase [EPO] and major basic protein [MBP] in eosinophils (see, forexample, Gleich G J. Mechanisms of eosinophil-associated inflammation. JAllergy Clin Immunol 2000; 105:651-663), lysozyme inmonocytes/macrophages (see, for example, Hoff T, Spencker T,Emmendoerffer A., Goppelt-Struebe M. Effects of glucocorticoids on theTPA-induced monocytic differentiation. J Leukoc Biol 1992; 52:173-182;and Balboa M A, Saez Y, Balsinde J. Calcium-independent phospholipase A2is required for lysozyme secretion in U937 promonocytes. J Immunol 2003;170:5276-5280), and granzyme in natural killer (NK) cells and cytotoxiclymphocytes (see, for example, Bochan M R, Goebel W S, Brahmi Z. Stablytransfected antisense granzyme B and perforin constructs inhibit humangranule-mediated lytic ability. Cell Immunol 1995; 164:234-239; Gong JH., Maki G, Klingemann H G. Characterization of a human cell line(NK-92) with phenotypical and functional characteristics of activatednatural killer cells. Leukemia 1994; 8:652-658; Maki G, Klingemann H G,Martinson J A, Tam Y K. Factors regulating the cytotoxic activity of thehuman natural killer cell line, NK-92. J Hematother Stem Cell Res 2001;10:369-383; and Takayama H, Trenn G, Sitkovsky M V. A novel cytotoxic Tlymphocyte activation assay. J Immunol Methods 1987; 104:183-190). Suchmediators are released at sites of injury and contribute to inflammationand tissue repair such as in the lung and elsewhere. It is known thatleukocytes release these granules via an exocytotic mechanism (see, forexample, Burgoyne R D, Morgan A. Secretory granule exocytosis. PhysiolRev 2003; 83:581-632; and Logan M R, Odemuyiwa S O, Moqbel R.Understanding exocytosis in immune and inflammatory cells: the molecularbasis of mediator secretion. J Allergy Clin Immunol 2003; 111: 923-932),but regulatory molecules and specific pathways involved in theexocytotic process have not been fully described.

Several exogenous stimuli can provoke degranulation of leukocytes via apathway that involves activation of protein kinase C and subsequentphosphorylation events (see, for example, Burgoyne R D, Morgan A.Secretory granule exocytosis. Physiol Rev 2003; 83:581-632; Logan M R,Odemuyiwa S O, Moqbel R. Understanding exocytosis in immune andinflammatory cells: the molecular basis of mediator secretion. J AllergyClin Immunol 2003; 111: 923-932; Smolen J E, Sandborg R R. Ca2+-inducedsecretion by electropermeabilized human neutrophils: the roles of Ca2+,nucleotides and protein kinase C. Biochim Biophys Acta 1990;1052:133-142; Niessen H W, Verhoeven A J. Role of proteinphosphorylation in the degranulation of electropermeabilized humanneutrophils. Biochim. Biophys. Acta 1994; 1223:267-273; and Naucler C,Grinstein S, Sundler R., Tapper H. Signaling to localized degranulationin neutrophils adherent to immune complexes. J Leukoc Biol 2002;71:701-710).

MARCKS protein (where MARCKS as used herein means “MyristoylatedAlanine-Rich C Kinase Substrate”), is a ubiquitous phosphorylationtarget of protein kinase C (PKC), and is highly expressed in leukocytes(see, for example, Aderem A A, Albert K A, Keum M M, Wang J K, GreengardP, Cohn Z A. Stimulus-dependent myristoylation of a major substrate forprotein kinase C. Nature 1988; 332:362-364; Thelen M, Rosen A, Nairn AC, Aderem A. Regulation by phosphorylation of reversible association ofa myristoylated protein kinase C substrate with the plasma membrane.Nature 1991; 351:320-322; and Hartwig J H, Thelen M, Rosen A, Janmey PA, Nairn A C, Aderem A. MARCKS is an actin filament crosslinking proteinregulated by protein kinase C and calcium-calmodulin. Nature 1992;356:618-622. MARCKS protein is mechanistically involved in a process ofexocytotic secretion of mucin by goblet cells that line respiratoryairways (see, for example, Li et al., J Biol Chem 2001; 276:40982-40990;and Singer et al., Nat Med 2004; 10:193-196). MARCKS is myristoylatedvia an amide bond at the N-terminal amino acid in the MARCKS protein'samino acid sequence at the alpha-amine position of the glycine whichresides at the N-terminus (i.e., at position 1) of amino acid sequence.In airway epithelial cells, the myristoylated N-terminal region ofMARCKS appears to be integral to the secretory process. By theN-terminus of the MARCKS protein is meant the MANS peptide whichcontains Myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1), which areL-amino acids. Additionally, the peptide fragments of the MANS peptidedisclosed herein, also preferably are composed of L-amino acids. Themechanism appears to involve binding of MARCKS, a myristoylated protein,to membranes of intracellular granules.

An N-terminal myristoylated peptide from the N-terminus of MARCKS hasbeen shown to block both mucin secretion and binding of MARCKS to mucingranule membranes in goblet cells (see, for example, Singer et al., NatMed 2004; 10:193-196). This peptide contains 24 amino acids of theMARCKS protein beginning with the N-terminal glycine of the MARCKSprotein which is myristoylated via an amide bond and is known asmyristoylated alpha-N-terminal sequence (MANS); i.e.,Myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1). Also Vergeres et al.,J. Biochem. 1998, 330; 5-11, discloses that the N-terminal glycineresidue of MARCKS proteins is myristoylated via a reaction catalyzed bymyristoyl CoA:protein N-myristoyl transferase (NMT).

In inflammatory diseases, such as asthma, COPD and chronic bronchitis;in genetic diseases such as cystic fibrosis; in allergic conditions(atopy, allergic inflammation); in bronchiectasis; and in a number ofacute, infectious respiratory illnesses such as pneumonia, rhinitis,influenza or the common cold, arthritis or auto-immune diseases,inflammatory cells are usually found in or migrate to areas of injury orinfection associated with inflammatory disease states, especially in orto respiratory passages or airways of patients suffering from suchdiseases. These inflammatory cells can contribute greatly to thepathology of diseases via tissue damage done by inflammatory mediatorsreleased from these cells. One example of such tissue damage ordestruction via this chronic inflammation occurs in cystic fibrosispatients where mediators released from neutrophils (e.g.,myeloperoxidase [MPO]) induce the desquamation of the airway epithelialtissue.

MARCKS, a protein of approximately 82 kD, has threeevolutionarily-conserved regions (Aderem et al., Nature 1988;332:362-364; Thelen et al., Nature 1991; 351:320-322; Hartwig et al.,Nature 1992; 356:618-622; Seykora et al., J Biol Chem 1996;271:18797-18802): an N-terminus, a phosphorylation site domain (or PSD),and a multiple homology 2 (MH2) domain. Human MARCKS cDNA and protein isknown and reported by Harlan et al., J. Biol. Chem. 1991, 266:14399(GenBank Accession No. M68956) and also by Sakai et al., Genomics 1992,14: 175. These sequences are also provided in a WO 00/50062, which isincorporated in its entirety by reference. The N-terminus, analpha-amino acid sequence comprising 24 amino acid residues with amyristic acid moiety attached via an amide bond to the N-terminalglycine residue is involved in binding of MARCKS to membranes in cells(Seykora et al., J Biol Chem 1996; 271:18797-18802) and possibly tocalmodulin (Matsubara et al., J Biol Chem 2003; 278:48898-48902). This24 amino acid sequence is known as the MANS peptide.

SUMMARY OF THE INVENTION

Involvement of MARCKS protein in release of inflammatory mediators fromthe granules of infiltrating leukocytes is relevant to inflammation indiseases in all tissues and organs, including lung diseasescharacterized by airway inflammation, such as asthma, COPD and cysticfibrosis. However, inflammation and mucus secretion in the airways aretwo separate and independent processes (Li et al., J Biol Chem 2001;276:40982-40990; Singer et al., Nat Med 2004; 10:193-196). While mucusproduction and secretion can be provoked by a number of factors,including mediators released by inflammatory cells, there is no knowndirect link whereby excess mucus causes inflammation.

In one aspect of this invention, the MANS peptide can play a role in thereducing the rate and/or amount of release of inflammatory mediatorsgranules or vesicles in inflammatory leukocytes.

In another aspect, peptides derived from the MARCKS N-terminus,especially from the 24 amino acid N-terminal sequence, i.e., activecontiguous peptide fragments derived from within the N-terminal 1-to-24amino acid sequence of MARCKS having a glycine at position 1, as well asN-terminal amides of such fragments, such as N-terminal acetic acidamides of such fragments, and/or as well as C-terminal amides of suchfragments, such as C-terminal amides of ammonia, can inhibit or reducethe rate and/or amount of release of inflammatory mediators frominflammatory leukocytes. Such inhibition or reduction in releasecomprises inhibition of a MARCKS-related release of inflammatorymediators from inflammatory leukocytes.

In another aspect, peptides derived from the MARCKS N-terminus,especially from the 1-to-24 amino acid N-terminal sequence, i.e., activecontiguous peptide fragments derived from within the N-terminal 1 to 24amino acid sequence of MARCKS having a glycine at position 1, as well asN-terminal amides of such fragments such as N-terminal acetic acidamides of such fragments, and as well as C-terminal amides of suchfragments such as C-terminal amides of ammonia, can inhibit the rate ofrelease and/or amount of release of inflammatory mediators such as thoseidentified herein in this invention, by inhibiting the process ofdegranulation in inflammatory leucocytes.

In another aspect, the MANS peptide and active fragments thereof, andactive amides of such fragments as described herein, can compete formembrane binding in inflammatory cells with native MARCKS protein toattenuate (lessen or reduce) MARCKS-related release of mediators ofinflammation from granules or vesicles containing such mediators ofinflammation in such inflammatory cells.

Leukocyte cell types and model cell types that secrete specific granulecontents in response to phorbol ester induced activation of PKC areuseful for in vitro demonstration of efficacy of peptides of thisinvention and of substituted peptides (e.g., alpha-N-amides, C-terminalamides and esters) of this invention.

The attenuation of release of membrane-bound inflammatory mediators bycompounds and compositions of this invention can be demonstrated usinghuman leukocyte cell lines. For example, neutrophils isolated from humanblood can be used to demonstrate attenuation or inhibition of release ofmyeloperoxidase (MPO). The human promyelocytic cell line HL-60 clone 15can be used to demonstrate attenuation of release or inhibition ofrelease or secretion of eosinophil peroxidase (EPO) by compounds andcompositions of this invention (see, for example, Fischkoff S A. Gradedincrease in probability of eosinophilic differentiation of HL-60promyelocytic leukemia cells induced by culture under alkalineconditions. Leuk Res 1988; 12:679-686; Rosenberg H F, Ackerman S J,Tenen D G. Human eosinophil cationic protein: molecular cloning of acytotoxin and helminthotoxin with ribonuclease activity. J Exp Med 1989;170:163-176; Tiffany H L, Li F, Rosenberg H F. Hyperglycosylation ofeosinophil ribonucleases in a promyelocytic leukemia cell line and indifferentiated peripheral blood progenitor cells. J Leukoc Biol 1995;58:49-54; and Badewa A P, Hudson C E, Heiman A S. Regulatory effects ofeotaxin, eotaxin-2, and eotaxin-3 on eosinophil degranulation andsuperoxide anion generation. Exp Biol Med 2002; 227:645-651). Themonocytic leukemia cell line U937 can be used to demonstrate attenuationof release or inhibition of release or secretion of lysozyme bycompounds and compositions of this invention (see, for example, Hoff T,Spencker T, Emmendoerffer A., Goppelt-Struebe M. Effects ofglucocorticoids on the TPA-induced monocytic differentiation. J LeukocBiol 1992; 52:173-182; Balboa M A, Saez Y, Balsinde J.Calcium-independent phospholipase A2 is required for lysozyme secretionin U937 promonocytes. J Immunol 2003; 170:5276-5280; and Sundstrom C,Nilsson K. Establishment and characterization of a human histiocyticlymphoma cell line (U-937). Int J Cancer 1976; 17:565-577). Thelymphocyte natural killer cell line NK-92 can be used to demonstrateattenuation or inhibition of release of granzyme by compounds andcompositions of this invention (see, for example, Gong J H., Maki G,Klingemann H G. Characterization of a human cell line (NK-92) withphenotypical and functional characteristics of activated natural killercells. Leukemia 1994; 8:652-658; Maki G, Klingemann H G, Martinson J A,Tam Y K. Factors regulating the cytotoxic activity of the human naturalkiller cell line, NK-92. J Hematother Stem Cell Res 2001; 10:369-383;and Takayama H, Trenn G, Sitkovsky M V. A novel cytotoxic T lymphocyteactivation assay. J Immunol Methods 1987; 104:183-190). In an in vitromethod to inhibit or attenuate the release of a mediator of inflammationsuch as those described herein, each of the cell types is preincubatedwith a peptide compound or peptide composition of this invention over arange of concentrations followed by incubation of these cells by astimulator of release of inflammatory mediators, such as phorbol ester.The percent of inhibition of release of a mediator of inflammation isdetermined as compared to the release of the mediator in the absence ofthe peptide compound or peptide composition, such as in aspecrophotometric readout of a concentration of the mediator released.

To demonstrate the importance of the relative amino acid sequencepositioning in the peptides of the invention, the relative ability toinhibit or reduce the amount of inflammatory mediator released by apeptide which is identical to the 24 amino acid sequence of the MARCKSprotein N-terminus region (i.e., the MANS—myristoylated alpha-N-terminalsequence peptide) was compared to the ability to inhibit or reduce theamount of inflammatory mediator released by a peptide containing thesame 24 amino acid residues present in MANS but which are sequenced in arandom order (i.e., an RNS peptide, otherwise referred to as a “RandomN-terminal sequence peptide”) with respect to the sequence order inMANS. In each of the cell types examined, the MANS peptide, but not theRNS peptide, attenuated release of inflammatory mediators in aconcentration-dependent manner over a time course of 0.5-3.0 hrs. Theseresults suggest that the relative amino acid sequence positioning in thepeptides of the invention which are in the order found in the MARCKSprotein, specifically its N-terminal region, and more specifically its24 amino acid residue N-terminal region are involved in at least oneintracellular pathway dealing with the inhibition of leukocytedegranulation.

The invention relates to a new use for the 24 amino acid peptidesequence, and to the alpha-N-terminal acetylated peptide sequence, themyristoylated polypeptide, also known as the MANS peptide, and to activefragments thereof, which active fragments can be selected from the groupof peptides having from 4 to 23 contiguous amino acid residues of theMANS peptide amino acid sequence, and which fragments may beN-terminal-myristoylated if they do not begin with the N-terminalglycine at position 1 in SEQ ID NO: 1, or which may beN-terminal-acylated with C2 to C12 acyl groups, includingN-terminal-acetylated, and/or C-terminal amidated with an NH2 group.

The invention also relates to a new method for blocking MARCKS-relatedcellular secretory processes, especially those that involve theMARCKS-related release of inflammatory mediators from inflammatorycells, whose stimulatory pathways involve the protein kinase C (PKC)substrate MARCKS protein and release of contents from intracellularvesicles or granules.

The present invention is directed to a method of inhibiting theexocytotic release of at least one inflammatory mediator from at leastone inflammatory cell comprising contacting the at least oneinflammatory cell, which cell comprises at least one inflammatorymediator contained within a vesicle inside the cell, with at least onepeptide selected from the group consisting of a MANS peptide and anactive fragment thereof as described herein in an effective amount toreduce the release of the inflammatory mediator from the inflammatorycell as compared to the release of the inflammatory mediator from thesame type of inflammatory cell that would occur in the absence of the atleast one peptide.

The present invention is further directed to a method of inhibiting therelease of at least one inflammatory mediator from at least oneinflammatory cell in a tissue or fluid of a subject comprising theadministration to the subject's tissue and/or fluid, which comprises atleast one inflammatory cell comprising at least one inflammatorymediator contained within a vesicle inside the cell, a therapeuticallyeffective amount of a pharmaceutical composition comprising at least onepeptide selected from the group consisting of a MANS peptide and anactive fragment thereof in a therapeutically effective amount to reducethe release of the inflammatory mediator from at least one inflammatorycell as compared to release of the inflammatory mediator from at leastone of the same type of inflammatory cell that would occur in theabsence of the at least one peptide. More specifically, inhibiting therelease of an inflammatory mediator comprises blocking or reducing therelease of an inflammatory mediator from the inflammatory cell.

More particularly, the present invention includes a method of reducinginflammation in a subject comprising the administration of atherapeutically effective amount of a pharmaceutical compositioncomprising a MANS peptide (i.e., N-myristoyl-GAQFSKTAAKGEAAAERPGEAAVA(SEQ ID NO: 1)) or an active fragment thereof. The active fragment is atleast four and preferably at least six amino acids in length. As usedherein, an “active fragment” of a MARCKS protein is one that affects(inhibits or reduces) MARCKS protein-mediated release, such as MARCKSprotein-mediated release of an inflammatory mediator. An active fragmentcan be selected from the group consisting of GAQFSKTAAKGEAAAERPGEAAV(SEQ ID NO: 2); GAQFSKTAAKGEAAAERPGEAA (SEQ ID NO: 4);GAQFSKTAAKGEAAAERPGEA (SEQ ID NO: 7); GAQFSKTAAKGEAAAERPGE (SEQ ID NO:11); GAQFSKTAAKGEAAAERPG (SEQ ID NO: 16); GAQFSKTAAKGEAAAERP (SEQ ID NO:22); GAQFSKTAAKGEAAAER (SEQ ID NO: 29); GAQFSKTAAKGEAAAE (SEQ ID NO:37); GAQFSKTAAKGEAAA (SEQ ID NO: 46); GAQFSKTAAKGEAA (SEQ ID NO: 56);GAQFSKTAAKGEA (SEQ ID NO: 67); GAQFSKTAAKGE (SEQ ID NO: 79); GAQFSKTAAKG(SEQ ID NO: 92); GAQFSKTAAK (SEQ ID NO: 106); GAQFSKTAA (SEQ ID NO:121); GAQFSKTA (SEQ ID NO: 137); GAQFSKT (SEQ ID NO: 154); GAQFSK (SEQID NO: 172); GAQFS (SEQ ID NO: 191) and GAQF (SEQ ID NO: 211). Thesepeptides, instead of containing a myristoyl moiety at the N-terminalamino acid, either contain no chemical moiety or a non-myristoylchemical moiety at the N-terminal amino acid and/or a chemical moiety atthe C-terminal amino acid, such as an N-terminal acetyl group and/or aC-terminal amide group as described herein. The presence of thehydrophobic N-terminal myristoyl moiety in the MANS peptides andN-terminal myristoylated fragments thereof can enhance theircompatibility with and presumably their permeability to plasmamembranes, and potentially enable the peptides to be taken up by cells.The hydrophobic insertion of a myristoyl group into a membrane lipidbilayer can provide a partition coefficient or apparent associationconstant with lipids of up to 10⁴ M⁻¹ or a unitary Gibbs free bindingenergy of about 8 kcal/mol (see, for example, Peitzsch, R. M., andMcLaughlin, S. 1993, Binding of acylated peptides and fatty acids tophospholipid vesicles: pertinence to myristoylated proteins.Biochemistry. 32: 10436-10443) which is sufficient, at least in part, topermit a partitioning of the MANS peptide and of myristoylated MANSpeptide fragments into the plasma membrane of a cell while additionalfunctional groups and their interactions within the MANS peptide (whichis myristoylated) and within myristoylated MANS peptide fragments canpotentiate their relative membrane permeabilities. The fragments caneach exhibit partition coefficients and membrane affinities that arerepresentative of their respective structure. The fragments can beprepared by methods of peptide synthesis known in the art, such as bysolid phase peptide synthesis (see, for example, the methods describedin Chan, Weng C. and White, Peter D. Eds., Fmoc Solid Phase PeptideSynthesis: A Practical Approach, Oxford University Press, New York, N.Y.(2000); and Lloyd-Williams, P. et al. Chemical Approaches to theSynthesis of Peptides and Proteins (1997)) and purified by methods knownin the art, such as by high pressure liquid chromatography. Molecularweight of each peptide can be confirmed by mass spectroscopy with eachshowing a peak with an appropriate molecular mass. Efficacy of theindividual peptides and of combinations of individual peptides (forexample, combinations of 2 of the peptides, combinations of 3 of thepeptides, combinations of 4 of the peptides) in the methods of thisdisclosure can be readily determined without undue experimentation usingthe procedures described in the examples disclosed herein. A preferredcombination will comprise two of the peptides; a preferred molar ratioof the peptides can be from 50:50 (i.e., 1:1) to 99.99 to 0.01, whichratio can be readily determined using the procedures described in theexamples disclosed herein.

Preferably the MANS peptide or active fragment thereof is contained in apharmaceutical composition which is useful to block inflammation. Thepresent invention also includes methods for inhibiting a cellularsecretory process in a subject comprising the administration of atherapeutically effective amount of a compound comprising a MANS peptideor an active fragment thereof, that inhibits an inflammatory mediator ina subject. The administration is generally selected from the groupconsisting of topical administration, parenteral administration, rectaladministration, pulmonary administration, inhalation and nasal or oraladministration, wherein pulmonary administration generally includeseither an aerosol, a dry powder inhaler, a metered dose inhaler, or anebulizer.

Administration of a composition comprising a degranulation-inhibitingamount of the MANS peptide or a degranulation-inhibiting amount of anactive fragment thereof, such as a pharmaceutical composition of theMANS peptide or an active fragment thereof, for human or animal useprovides the MANS peptide or active fragment thereof at least to thesite in or on a tissue or to a fluid-containing layer in contact withthe surface of a tissue where an inflammatory granulocytic cell residesor into which an inflammatory granulocytic cell will invade, thusenabling the MANS peptide or an active fragment thereof to contact theinflammatory granulocytic cell. In one aspect, administration of such acomposition can be made at the first onset or first detection ofinflammation or first perception of inflammation by the human or animalor at the first perceptible change in the level of inflammation in ahuman or animal to reduce the amount of inflammation that wouldotherwise occur in the absence of the MANS peptide or active fragmentthereof. In another aspect, administration can be made during an ongoinginflammation of a tissue in the human or animal to reduce the amount ofadditional inflammation that would otherwise occur in the absence of theMANS peptide or active fragment thereof. While the amount and frequencyof dose can be determined by clinical evaluation and be a function ofthe disease or source of inflammation and the extent of tissue involvedand the age and size of the patient, it is anticipated that dosing of apharmaceutical composition can be repeated after 3 to 8 hours,preferably after 6 to 8 hours after the first administration of thepharmaceutical composition.

The present invention also includes methods of reducing inflammation ina subject comprising the administration of a therapeutically effectiveamount of a compound that inhibits the MARCKS-related release ofinflammatory mediators, whereby the release of at least one inflammatorymediator in the subject is reduced compared to that which would occur inthe absence of said treatment. As used herein “reducing” generally meansa lessening of the effects of inflammation. Preferably, release ofinflammatory mediators are inhibited or blocked by the methodsdisclosed.

Another embodiment of the present invention includes methods of reducinginflammation in a subject comprising administering a therapeuticallyeffective amount of a compound that inhibits the MARCKS-related releaseof inflammatory mediators, whereby the inflammation in the subject isreduced compared to that which would occur in the absence of saidtreatment. The present invention also discloses methods of reducing orinhibiting inflammation in a subject comprising the administration of atherapeutically effective amount of a MANS peptide or an active fragmentthereof effective to inhibit an inflammatory mediator at theinflammation site. The term “inhibiting” means a reduction in the amountof inflammatory mediator secretion. The term “completely inhibiting”means a reduction to zero in the amount of inflammatory mediatorsecretion. Again, as stated above, the active fragment is at least fourand preferably at least six amino acids in length. The term “exocytoticprocess” means exocytosis, i.e., a process of cellular secretion orexcretion in which substances contained in a vesicle, which vesicleresides inside a cell, are discharged from the cell by fusion of thevesicular membrane of the vesicle with the outer cell membrane.“Degranulation” means the release of cellular granule contents. The term“degranulation-inhibiting” means a reduction in the release of theinflammatory mediators contained within the granules of the inflammatorycell. Thus, a degranulation-inhibiting amount of the MANS peptide and/oran active fragment thereof is the amount of these peptides that issufficient to reduce the release of the inflammatory mediators containedin the granules as compared to release in the absence of the samepeptide.

In the reference peptide, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1), atthe N-terminal position of the reference peptide, G is at position 1;adjacent to G at position 1 is A at position 2; adjacent to A atposition 2 is Q at position 3; adjacent to Q at position 3 is F atposition 4; adjacent to F at position 4 is S at position 5; adjacent toS at position 5 is K at position 6; adjacent to K at position 6 is T atposition 7; adjacent to T at position 7 is A at position 8; adjacent toA at position 8 is A at position 9; adjacent to A at position 9 is K atposition 10; adjacent to K at position 10 is G at position 11; adjacentto G at position 11 is E at position 12; adjacent to E at position 12 isA at position 13; adjacent to A at position 13 is A at position 14;adjacent to A at position 14 is A at position 15; adjacent to A atposition 15 is E at position 16; adjacent to E at position 16 is R atposition 17; adjacent to R at position 17 is P at position 18; adjacentto P at position 18 is G at position 19; adjacent to G at position 19 isE at position 20; adjacent to E at position 20 is A at position 21;adjacent to A at position 21 is A at position 22; adjacent to A atposition 22 is V at position 23; and adjacent to V at position 23 is Aat position 24, wherein position 24 is the C-terminal position of thereference peptide.

A “variant” of a reference peptide or a variant of a 4 to 23 amino acidsegment of a reference peptide is a peptide which has an amino acidsequence which differs from the amino acid sequence of the referencepeptide or from the amino acid sequence of the segment of the referencepeptide, respectively, in at least one amino acid position in thereference peptide or reference peptide segment amino acid sequence,respectively, but which retains mucin- or mucus-inhibiting activity,which activity is typically between 0.1 to 10 times the activity of thereference peptide or segment, respectively, preferably between 0.2 to 6times the activity of the reference peptide or segment, respectively,more preferably between 0.3 to 5 times the activity of the referencepeptide or segment, respectively. A “variant” of a reference amino acidsequence or a variant of a 4 to 23 amino acid segment of a referenceamino acid sequence is an amino acid sequence that differs by at leastone amino acid from the reference amino acid sequence or from thesegment of the reference amino acid sequence, respectively, but has anamino acid sequence of a peptide that retains mucin- or mucus-inhibitingactivity of the peptide or segment, respectively, encoded by thereference amino acid sequence, which activity is typically between 0.1to 10 times the activity of the peptide or segment, respectively, of thereference sequence, preferably between 0.2 to 6 times the activity ofthe peptide or segment of the reference sequence, respectively, morepreferably between 0.3 to 5 times the activity of the peptide or segmentof the reference sequence, respectively. A substitution variant peptideor a substitution variant amino acid sequence may vary (i.e., differ)from a reference peptide or reference amino acid sequence by one or moreamino acid substitutions in the reference amino acid sequence; adeletion variant peptide or a deletion variant amino acid sequence mayvary (i.e., differ) from a reference peptide or reference amino acidsequence by one or more amino acid deletions in the reference amino acidsequence; and an addition variant peptide or an addition variant aminoacid sequence may vary (i.e., differ) from a reference peptide sequenceor reference amino acid sequence by one or more amino acid additions inthe reference sequence. A variant peptide or variant amino acid sequencecan result from a substitution of one or more amino acids (e.g.,substitution of at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acids) in areference sequence, or can result from a deletion of one or more aminoacids (e.g., deletion of at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acids)in a reference sequence, or can result from an addition of one or moreamino acids (e.g., addition of at least 1, 2, 3, 4, 5, 6, 7, or 8 aminoacids) in a reference sequence, or a combination thereof in any order. Asubstitution variant 4 to 23 amino acid peptide segment or asubstitution variant 4 to 23 amino acid segment sequence may vary (i.e.,differ) from a reference 4 to 23 amino acid peptide segment or reference4 to 23 amino acid segment sequence by one or more amino acidsubstitutions in the reference amino acid segment sequence; a deletionvariant 4 to 23 amino acid peptide segment or a 4 to 22 amino aciddeletion variant amino acid segment sequence may vary (i.e., differ)from a 5 to 23 reference peptide segment or a 5 to 23 amino acidreference amino acid segment sequence by one or more amino aciddeletions in the reference amino acid segment sequence; and an 4 to 23amino acid addition variant peptide or a 4 to 23 amino acid additionvariant amino acid sequence may vary (i.e., differ) from a 4 to 22 aminoacid reference peptide sequence or a 4 to 22 amino acid reference aminoacid sequence by one or more amino acid additions in the referencesequence. A 4 to 23 amino acid variant peptide or a 4 to 23 amino acidvariant amino acid sequence can result from a substitution of one ormore amino acids (e.g., substitution of at least 1, 2, 3, 4, 5, 6, 7, 8amino acids) in a 4 to 23 amino acid segment of a reference amino acidsequence, or can result from a deletion of one or more amino acids(e.g., deletion of at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acids) in arespectively larger reference amino acid sequence, or can result from anaddition of one or more amino acids (e.g., addition of at least 1, 2, 3,4, 5, 6, 7, or 8 amino acids) in a respectively smaller reference aminoacid sequence, or from a combination thereof. Preferably, a variantpeptide or amino acid sequence varies from a reference peptide or from asegment of a reference peptide or from a reference amino acid sequenceor from a segment of a reference amino acid sequence, respectively, byless than 10 amino acid substitutions, deletions, and/or additions; morepreferably less than 8 amino acid substitutions, deletions, and/oradditions; even more preferably less than 6 amino acid substitutions,deletions, and/or additions; and even more preferably less than 5 aminoacid substitutions, deletions, and/or additions; and yet even morepreferably less than 4 amino acid substitutions, deletions, and/oradditions. Most preferably the variant amino acid sequence differs froma reference peptide or segment amino acid sequence by one or two orthree amino acids.

“Sequence identity” means, with respect to amino acid sequences of twopeptides, the number of positions with identical amino acids divided bythe number of amino acids in the shorter of the two sequences.

“Substantially identical” means, with respect to comparison of the aminoacid sequences of two peptides or comparison of the amino acid sequencesof two peptide segments (e.g. segments of a reference peptide amino acidsequence), that the amino acid sequence of the peptides or segments ofpeptides have at least 75% sequence identity, preferably at least 80%sequence identity, more preferably at least 90% sequence identity, andmost preferably at least 95% sequence identity.

The term “peptide” as used herein includes the peptide as well aspharmaceutically acceptable salts of the peptide.

An “isolated” peptide, as used herein, means a naturally-occurringpeptide that has been separated or substantially separated from thecellular components (e.g., nucleic acids and other peptides) thatnaturally accompany it by purification, recombinant synthesis, orchemical synthesis, and also encompasses non-naturally-occurringrecombinantly or chemically synthesized peptides that have been purifiedor substantially purified from cellular components, biologicalmaterials, chemical precursors, or other chemicals.

The following three-letter and one-letter amino acid abbreviations areused throughout the text: Alanine: (Ala) A; Arginine: (Arg) R;Asparagine: (Asn) N; Aspartic acid: (Asp) D; Cysteine: (Cys) C;Glutamine: (Gin) Q; Glutamic acid: (Glu) E; Glycine: (Gly) G; Histidine:(His) H; Isoleucine: (Ile) I; Leucine: (Leu) L; Lysine: (Lys) K;Methionine: (Met) M; Phenylalanine: (Phe) F; Proline: (Pro) P; Serine:(Ser) S; Threonine: (Thr) T; Tryptophan: (Trp) W; Tyrosine: (Tyr) Y;Valine: (Val) V. Additional three letter symbols of amino acids usefulherein include, in brackets, (Hyp) for hydroxyproline, (Nle) fornorleucine, (Orn) for ornithine, (Pyr) for pyroglutamic acid and (Sar)for sarcosine. By convention, the amino (or N-terminal) end of a peptideappears at the left end of a written amino acid sequence of the peptideand the carboxy (or C-terminal) end appears at the right end of awritten amino acid sequence. The amino acid sequence of a peptide can bewritten in single letter symbols to represent the amino acids which arecovalently linked by peptide amide bonds in the peptide.

Active fragments of the MANS peptide can be useful in the prevention orreduction in amount of inflammation in a tissue in an animal caused byinflammatory mediators. Active fragments of the MANS peptide also can beuseful in the prevention or reduction in amount of tissue damage in ananimal produced or caused by inflammatory mediators. An active fragmentof the MANS peptide is composed of at least 4 contiguous amino acids andno more than 23 contiguous amino acids of the MANS peptide (SEQ ID NO:1). The term “active fragment” within the context of the presentinvention is intended to encompass those fragments of the MANS peptidesthat are capable of preventing or reducing the release of inflammatorymediators from an inflammatory cell. The reduction of release ofinflammatory mediators by the active fragments can range from at least5% to at least 99% reduction as compared to a reference peptide, such asMANS peptide.

Table 1 contains a list of amino acid sequences in single letterabbreviation format together with a respectively corresponding peptidenumber and SEQ ID NO. The reference peptide amino acid sequence (MANSpeptide) is listed as peptide 1. Amino acid sequences of peptides of theinvention having an amino acid sequence of from 4 to 23 contiguous aminoacids of the reference amino acid sequence are listed as peptides 2 to231, together with the amino acid sequence of a random N-terminalsequence (RNS) comprising amino acids of the MANS peptide as peptide232. Amino acid sequences of representative variants of amino acidsequences of peptides of the invention as described herein and are alsolisted as peptides 233 to 245 and 247 to 251. This variant peptideslisted are not intended to be a limiting group of peptides, but arepresented only to serve as representative examples of variant peptidesof the invention. Also presented is a representative reverse amino acidsequence (peptide 246) and a representative random amino acid sequenceof peptide (peptide 232) of the invention. The reverse and random aminoacid sequences in the table are not intended to be representative of theinvention.

Table 1 contains a listing of peptides of this invention and theirrespective amino acid sequences and corresponding SEQ ID NOS.

TABLE 1 Peptides and Amino Acid Sequences Peptide Sequence No. SequenceID No. peptide 1 GAQFSKTAAKGEAAAERPGEAAVA SEQ ID NO. 1 peptide 2GAQFSKTAAKGEAAAERPGEAAV SEQ ID NO. 2 peptide 3 AQFSKTAAKGEAAAERPGEAAVASEQ ID NO. 3 peptide 4 GAQFSKTAAKGEAAAERPGEAA SEQ ID NO. 4 peptide 5AQFSKTAAKGEAAAERPGEAAV SEQ ID NO. 5 peptide 6 QFSKTAAKGEAAAERPGEAAVASEQ ID NO. 6 peptide 7 GAQFSKTAAKGEAAAERPGEA SEQ ID NO. 7 peptide 8AQFSKTAAKGEAAAERPGEAA SEQ ID NO. 8 peptide 9 QFSKTAAKGEAAAERPGEAAVSEQ ID NO. 9 peptide 10 FSKTAAKGEAAAERPGEAAVA SEQ ID NO. 10 peptide 11GAQFSKTAAKGEAAAERPGE SEQ ID NO. 11 peptide 12 AQFSKTAAKGEAAAERPGEASEQ ID NO. 12 peptide 13 QFSKTAAKGEAAAERPGEAA SEQ ID NO. 13 peptide 14FSKTAAKGEAAAERPGEAAV SEQ ID NO. 14 peptide 15 SKTAAKGEAAAERPGEAAVASEQ ID NO. 15 peptide 16 GAQFSKTAAKGEAAAERPG SEQ ID NO. 16 peptide 17AQFSKTAAKGEAAAERPGE SEQ ID NO. 17 peptide 18 QFSKTAAKGEAAAERPGEASEQ ID NO. 18 peptide 19 FSKTAAKGEAAAERPGEAA SEQ ID NO. 19 peptide 20SKTAAKGEAAAERPGEAAV SEQ ID NO. 20 peptide 21 KTAAKGEAAAERPGEAAVASEQ ID NO. 21 peptide 22 GAQFSKTAAKGEAAAERP SEQ ID NO. 22 peptide 23AQFSKTAAKGEAAAERPG SEQ ID NO. 23 peptide 24 QFSKTAAKGEAAAERPGESEQ ID NO. 24 peptide 25 FSKTAAKGEAAAERPGEA SEQ ID NO. 25 peptide 26SKTAAKGEAAAERPGEAA SEQ ID NO. 26 peptide 27 KTAAKGEAAAERPGEAAVSEQ ID NO. 27 peptide 28 TAAKGEAAAERPGEAAVA SEQ ID NO. 28 peptide 29GAQFSKTAAKGEAAAER SEQ ID NO. 29 peptide 30 AQFSKTAAKGEAAAERPSEQ ID NO. 30 peptide 31 QFSKTAAKGEAAAERPG SEQ ID NO. 31 peptide 32FSKTAAKGEAAAERPGE SEQ ID NO. 32 peptide 33 SKTAAKGEAAAERPGEASEQ ID NO. 33 peptide 34 KTAAKGEAAAERPGEAA SEQ ID NO. 34 peptide 35TAAKGEAAAERPGEAAV SEQ ID NO. 35 peptide 36 AAKGEAAAERPGEAAVASEQ ID NO. 36 peptide 37 GAQFSKTAAKGEAAAE SEQ ID NO. 37 peptide 38AQFSKTAAKGEAAAER SEQ ID NO. 38 peptide 39 QFSKTAAKGEAAAERP SEQ ID NO. 39peptide 40 FSKTAAKGEAAAERPG SEQ ID NO. 40 peptide 41 SKTAAKGEAAAERPGESEQ ID NO. 41 peptide 42 KTAAKGEAAAERPGEA SEQ ID NO. 42 peptide 43TAAKGEAAAERPGEAA SEQ ID NO. 43 peptide 44 AAKGEAAAERPGEAAV SEQ ID NO. 44peptide 45 AKGEAAAERPGEAAVA SEQ ID NO. 45 peptide 46 GAQFSKTAAKGEAAASEQ ID NO. 46 peptide 47 AQFSKTAAKGEAAAE SEQ ID NO. 47 peptide 48QFSKTAAKGEAAAER SEQ ID NO. 48 peptide 49 FSKTAAKGEAAAERP SEQ ID NO. 49peptide 50 SKTAAKGEAAAERPG SEQ ID NO. 50 peptide 51 KTAAKGEAAAERPGESEQ ID NO. 51 peptide 52 TAAKGEAAAERPGEA SEQ ID NO. 52 peptide 53AAKGEAAAERPGEAA SEQ ID NO. 53 peptide 54 AKGEAAAERPGEAAV SEQ ID NO. 54peptide 55 KGEAAAERPGEAAVA SEQ ID NO. 55 peptide 56 GAQFSKTAAKGEAASEQ ID NO. 56 peptide 57 AQFSKTAAKGEAAA SEQ ID NO. 57 peptide 58QFSKTAAKGEAAAE SEQ ID NO. 58 peptide 59 FSKTAAKGEAAAER SEQ ID NO. 59peptide 60 SKTAAKGEAAAERP SEQ ID NO. 60 peptide 61 KTAAKGEAAAERPGSEQ ID NO. 61 peptide 62 TAAKGEAAAERPGE SEQ ID NO. 62 peptide 63AAKGEAAAERPGEA SEQ ID NO. 63 peptide 64 AKGEAAAERPGEAA SEQ ID NO. 64peptide 65 KGEAAAERPGEAAV SEQ ID NO. 65 peptide 66 GEAAAERPGEAAVASEQ ID NO. 66 peptide 67 GAQFSKTAAKGEA SEQ ID NO. 67 peptide 68AQFSKTAAKGEAA SEQ ID NO. 68 peptide 69 QFSKTAAKGEAAA SEQ ID NO. 69peptide 70 FSKTAAKGEAAAE SEQ ID NO. 70 peptide 71 SKTAAKGEAAAERSEQ ID NO. 71 peptide 72 KTAAKGEAAAERP SEQ ID NO. 72 peptide 73TAAKGEAAAERPG SEQ ID NO. 73 peptide 74 AAKGEAAAERPGE SEQ ID NO. 74peptide 75 AKGEAAAERPGEA SEQ ID NO. 75 peptide 76 KGEAAAERPGEAASEQ ID NO. 76 peptide 77 GEAAAERPGEAAV SEQ ID NO. 77 peptide 78EAAAERPGEAAVA SEQ ID NO. 78 peptide 79 GAQFSKTAAKGE SEQ ID NO. 79peptide 80 AQFSKTAAKGEA SEQ ID NO. 80 peptide 81 QFSKTAAKGEAASEQ ID NO. 81 peptide 82 FSKTAAKGEAAA SEQ ID NO. 82 peptide 83SKTAAKGEAAAE SEQ ID NO. 83 peptide 84 KTAAKGEAAAER SEQ ID NO. 84peptide 85 TAAKGEAAAERP SEQ ID NO. 85 peptide 86 AAKGEAAAERPGSEQ ID NO. 86 peptide 87 AKGEAAAERPGE SEQ ID NO. 87 peptide 88KGEAAAERPGEA SEQ ID NO. 88 peptide 89 GEAAAERPGEAA SEQ ID NO. 89peptide 90 EAAAERPGEAAV SEQ ID NO. 90 peptide 91 AAAERPGEAAVASEQ ID NO. 91 peptide 92 GAQFSKTAAKG SEQ ID NO. 92 peptide 93AQFSKTAAKGE SEQ ID NO. 93 peptide 94 QFSKTAAKGEA SEQ ID NO. 94peptide 95 FSKTAAKGEAA SEQ ID NO. 95 peptide 96 SKTAAKGEAAASEQ ID NO. 96 peptide 97 KTAAKGEAAAE SEQ ID NO. 97 peptide 98TAAKGEAAAER SEQ ID NO. 98 peptide 99 AAKGEAAAERP SEQ ID NO. 99 peptide AKGEAAAERPG SEQ ID NO. 100 100 peptide  KGEAAAERPGE SEQ ID NO. 101 101peptide  GEAAAERPGEA SEQ ID NO. 102 102 peptide  EAAAERPGEAASEQ ID NO. 103 103 peptide  AAAERPGEAAV SEQ ID NO. 104 104 peptide AAERPGEAAVA SEQ ID NO. 105 105 peptide  GAQFSKTAAK SEQ ID NO. 106 106peptide  AQFSKTAAKG SEQ ID NO. 107 107 peptide  QFSKTAAKGESEQ ID NO. 108 108 peptide  FSKTAAKGEA SEQ ID NO. 109 109 peptide SKTAAKGEAA SEQ ID NO. 110 110 peptide  KTAAKGEAAA SEQ ID NO. 111 111peptide  TAAKGEAAAE SEQ ID NO. 112 112 peptide  AAKGEAAAERSEQ ID NO. 113 113 peptide  AKGEAAAERP SEQ ID NO. 114 114 peptide KGEAAAERPG SEQ ID NO. 115 115 peptide  GEAAAERPGE SEQ ID NO. 116 116peptide  EAAAERPGEA SEQ ID NO. 117 117 peptide  AAAERPGEAASEQ ID NO. 118 118 peptide  AAERPGEAAV SEQ ID NO. 119 119 peptide AERPGEAAVA SEQ ID NO. 120 120 peptide  GAQFSKTAA SEQ ID NO. 121 121peptide  AQFSKTAAK SEQ ID NO. 122 122 peptide  QFSKTAAKG SEQ ID NO. 123123 peptide  FSKTAAKGE SEQ ID NO. 124 124 peptide  SKTAAKGEASEQ ID NO. 125 125 peptide  KTAAKGEAA SEQ ID NO. 126 126 peptide TAAKGEAAA SEQ ID NO. 127 127 peptide  AAKGEAAAE SEQ ID NO. 128 128peptide  AKGEAAAER SEQ ID NO. 129 129 peptide  KGEAAAERP SEQ ID NO. 130130 peptide  GEAAAERPG SEQ ID NO. 131 131 peptide  EAAAERPGESEQ ID NO. 132 132 peptide  AAAERPGEA SEQ ID NO. 133 133 peptide AAERPGEAA SEQ ID NO. 134 134 peptide  AERPGEAAV SEQ ID NO. 135 135peptide  ERPGEAAVA SEQ ID NO. 136 136 peptide  GAQFSKTA SEQ ID NO. 137137 peptide  AQFSKTAA SEQ ID NO. 138 138 peptide  QFSKTAAKSEQ ID NO. 139 139 peptide  FSKTAAKG SEQ ID NO. 140 140 peptide SKTAAKGE SEQ ID NO. 141 141 peptide  KTAAKGEA SEQ ID NO. 142 142peptide  TAAKGEAA SEQ ID NO. 143 143 peptide  AAKGEAAA SEQ ID NO. 144144 peptide  AKGEAAAE SEQ ID NO. 145 145 peptide  KGEAAAERSEQ ID NO. 146 146 peptide  GEAAAERP SEQ ID NO. 147 147 peptide EAAAERPG SEQ ID NO. 148 148 peptide  AAAERPGE SEQ ID NO. 149 149peptide  AAERPGEA SEQ ID NO. 150 150 peptide  AERPGEAA SEQ ID NO. 151151 peptide  ERPGEAAV SEQ ID NO. 152 152 peptide  RPGEAAVASEQ ID NO. 153 153 peptide  GAQFSKT SEQ ID NO. 154 154 peptide  AQFSKTASEQ ID NO. 155 155 peptide  QFSKTAA SEQ ID NO. 156 156 peptide  FSKTAAKSEQ ID NO. 157 157 peptide  SKTAAKG SEQ ID NO. 158 158 peptide  KTAAKGESEQ ID NO. 159 159 peptide  TAAKGEA SEQ ID NO. 160 160 peptide  AAKGEAASEQ ID NO. 161 161 peptide  AKGEAAA SEQ ID NO. 162 162 peptide  KGEAAAESEQ ID NO. 163 163 peptide  GEAAAER SEQ ID NO. 164 164 peptide  EAAAERPSEQ ID NO. 165 165 peptide  AAAERPG SEQ ID NO. 166 166 peptide  AAERPGESEQ ID NO. 167 167 peptide  AERPGEA SEQ ID NO. 168 168 peptide  ERPGEAASEQ ID NO. 169 169 peptide  RPGEAAV SEQ ID NO. 170 170 peptide  PGEAAVASEQ ID NO. 171 171 peptide  GAQFSK SEQ ID NO. 172 172 peptide  AQFSKTSEQ ID NO. 173 173 peptide  QFSKTA SEQ ID NO. 174 174 peptide  FSKTAASEQ ID NO. 175 175 peptide  SKTAAK SEQ ID NO. 176 176 peptide  KTAAKGSEQ ID NO. 177 177 peptide  TAAKGE SEQ ID NO. 178 178 peptide  AAKGEASEQ ID NO. 179 179 peptide  AKGEAA SEQ ID NO. 180 180 peptide  KGEAAASEQ ID NO. 181 181 peptide  GEAAAE SEQ ID NO. 182 182 peptide  EAAAERSEQ ID NO. 183 183 peptide  AAAERP SEQ ID NO. 184 184 peptide  AAERPGSEQ ID NO. 185 185 peptide  AERPGE SEQ ID NO. 186 186 peptide  ERPGEASEQ ID NO. 187 187 peptide  RPGEAA SEQ ID NO. 188 188 peptide  PGEAAVSEQ ID NO. 189 189 peptide  GEAAVA SEQ ID NO. 190 190 peptide  GAQFSSEQ ID NO. 191 191 peptide  AQFSK SEQ ID NO. 192 192 peptide  QFSKTSEQ ID NO. 193 193 peptide  FSKTA SEQ ID NO. 194 194 peptide  SKTAASEQ ID NO. 195 195 peptide  KTAAK SEQ ID NO. 196 196 peptide  TAAKGSEQ ID NO. 197 197 peptide  AAKGE SEQ ID NO. 198 198 peptide  AKGEASEQ ID NO. 199 199 peptide  KGEAA SEQ ID NO. 200 200 peptide  GEAAASEQ ID NO. 201 201 peptide  EAAAE SEQ ID NO. 202 202 peptide  AAAERSEQ ID NO. 203 203 peptide  AAERP SEQ ID NO. 204 204 peptide  AERPGSEQ ID NO. 205 205 peptide  ERPGE SEQ ID NO. 206 206 peptide  RPGEASEQ ID NO. 207 207 peptide  PGEAA SEQ ID NO. 208 208 peptide  GEAAVSEQ ID NO. 209 209 peptide  EAAVA SEQ ID NO. 210 210 peptide  GAQFSEQ ID NO. 211 211 peptide  AQFS SEQ ID NO. 212 212 peptide  QFSKSEQ ID NO. 213 213 peptide  FSKT SEQ ID NO. 214 214 peptide  SKTASEQ ID NO. 215 215 peptide  KTAA SEQ ID NO. 216 216 peptide  TAAKSEQ ID NO. 217 217 peptide  AAKG SEQ ID NO. 218 218 peptide  AKGESEQ ID NO. 219 219 peptide  KGEA SEQ ID NO. 220 220 peptide  GEAASEQ ID NO. 221 221 peptide  EAAA SEQ ID NO. 222 222 peptide  AAAESEQ ID NO. 223 223 peptide  AAER SEQ ID NO. 224 224 peptide  AERPSEQ ID NO. 225 225 peptide  ERPG SEQ ID NO. 226 226 peptide  RPGESEQ ID NO. 227 227 peptide  PGEA SEQ ID NO. 228 228 peptide  GEAASEQ ID NO. 229 229 peptide  EAAV SEQ ID NO. 230 230 peptide  AAVASEQ ID NO. 231 231 peptide  GTAPAAEGAGAEVKRASAEAKQ SEQ ID NO. 232 232 AFpeptide  GKQFSKTAAKGE SEQ ID NO. 233 233 peptide  GAQFSKTKAKGESEQ ID NO. 234 234 peptide  GKQFSKTKAKGE SEQ ID NO. 235 235 peptide GAQASKTAAK SEQ ID NO. 236 236 peptide  GAQASKTAAKGE SEQ ID NO. 237 237peptide  GAEFSKTAAKGE SEQ ID NO. 238 238 peptide  GAQFSKTAAAGESEQ ID NO. 239 239 peptide  GAQFSKTAAKAE SEQ ID NO. 240 240 peptide GAQFSKTAAKGA SEQ ID NO. 241 241 peptide  AAQFSKTAAK SEQ ID NO. 242 242peptide  GAAFSKTAAK SEQ ID NO. 243 243 peptide  GAQFAKTAAKSEQ ID NO. 244 244 peptide  GAQFSATAAK SEQ ID NO. 245 245 peptide KAATKSFQAG SEQ ID NO. 246 246 peptide  GAQFSKAAAK SEQ ID NO. 247 247peptide  GAQFSKTAAA SEQ ID NO. 248 248 peptide  GAQFSATAAASEQ ID NO. 249 249 peptide  GAQASKTA SEQ ID NO. 250 250 peptide  AAGESEQ ID NO. 251 251 peptide  GKASQFAKTA SEQ ID NO. 252 252

An amino acid sequence of a peptide listed in Table 1 can be chemicallymodified. For example, if an amino acid sequence of a peptide listed inTable 1 is chemically modified at the N-terminal amine to form an amidewith a carboxylic acid, the resulting peptide is sometimes referred toherein by a combination of an identifier for the carboxylic acid as aprefix linked by a hyphen to the peptide number. For example, withrespect to peptide 79 as an example, an N-terminal myristoylated peptide79 may sometimes be referred to herein as “myristoylated-peptide 79” or“myr-peptide 79”; an N-terminal acetylated peptide 79 may sometimes bereferred to herein as “acetyl-peptide 79” or “Ac-peptide 79”. A cyclicversion of peptide 79 may be referred to as “cyclic-peptide 79” or“cyc-peptide 79”. Also, for example, if an amino acid sequence of apeptide listed in Table 1 is chemically modified at the C-terminalcarboxylic group, for example by an amine such as ammonia to form aC-terminal amide, the resulting peptide is sometimes referred to hereinby a combination of an identifier for the amine residue as a suffixlinked by a hyphen to the peptide number. Thus, for example, aC-terminal amide of peptide 79 can be sometimes referred to as“peptide-NH2”. When the N-terminal amine of the peptide (e.g., peptide79) is chemically modified by, for example, a myristoyl group and theC-terminal carboxylic group is chemically modified by, for example, anammonia group to form an amide as above, the resulting peptide can besometimes referred to, using both prefix and suffix notation, as“myr-peptide 79-NH2”.

The invention involves peptides having amino acid sequences comprisingless than 24 amino acids with amino acid sequences related to the aminoacid sequence of MANS peptide (i.e., the MANS peptide ismyristoyl-peptide 1 and the reference 24-amino acid sequence of the MANSpeptide is peptide 1). The peptides of the current invention consist ofamino acid sequences containing less than 24 amino acids, and mayconsist of from 8 to 14, from 10 to 12, from 9 to 14, from 9 to 13, from10 to 13, from 10 to 14, at least 9, at least 10, or the like aminoacids. The peptides are typically straight chains, but may be cyclicpeptides as well. In addition, the peptides may be isolated peptides.

With respect to peptide 1 (SEQ ID NO: 1), the reference 24 amino acidsequence, a segment of 23 continuous amino acids of the reference aminoacid sequence is sometimes referred to herein as a 23-mer. Analogously,a segment of 22 continuous amino acids of the reference sequence issometimes referred to herein as a 22-mer; a 21 amino acid sequence as a21-mer; a 20 amino acid sequence as a 20-mer; a 19 amino acid sequenceas a 19-mer; an 18 amino acid sequence as an 18-mer; a 17 amino acidsequence as a 17-mer; a 16 amino acid sequence as a 16-mer; a 15 aminoacid sequence as a 15-mer; a 14 amino acid sequence as a 14-mer; a 13amino acid sequence as a 13-mer; a 12 amino acid sequence as a 12-mer;an 11 amino acid sequence as an 11-mer; a 10 amino acid sequence as a10-mer; a 9 amino acid sequence as a 9-mer; an 8 amino acid sequence asan 8-mer; a 7 amino acid sequence as a 7-mer; a 6 amino acid sequence asa 6-mer; a 5 amino acid sequence as a 5-mer; and a 4 amino acid sequenceas a 4-mer. In one aspect, any of these “4- to 23-mer” amino acidsequences, which are themselves peptides (sometimes herein denoted asH2N-peptide-COOH), can be independently chemically modified, forexample, by chemical modification, which chemical modification can beselected from the group consisting of (i) amide formation at theN-terminal amine group (H2N-peptide-) such as with, for example, a C1 orpreferably with a C2 (acetic acid) to C22 carboxylic acid; (ii) amideformation at the C-terminal carboxylic group (-peptide-COOH) such aswith, for example, ammonia or with a C1 to C22 primary or secondaryamine; and (iii) a combination of thereof.

The peptides have an amino acid sequence selected from the groupconsisting of (a) an amino acid sequence having from 4 to 23 contiguousamino acids of the reference sequence, peptide 1; (b) a sequencesubstantially similar to the amino acid sequence defined in (a); and (c)a variant of the amino acid sequence defined in (a), which variant isselected from the group consisting of a substitution variant, a deletionvariant, an addition variant, and combinations thereof. In someembodiments, the peptides have an amino acid sequence selected from thegroup consisting of: (a) an amino acid sequence having from 8 to 14contiguous amino acids of the reference sequence, peptide 1; (b) anamino acid sequence substantially identical to the sequence defined in(a); and (c) a variant of the amino acid sequence defined in (a), whichvariant is selected from the group consisting of a substitution variant,a deletion variant, an addition variant, and combinations thereof. Inyet other embodiments, the peptides have an amino acid sequence selectedfrom the group consisting of: (a) an amino acid sequence having from 10to 12 contiguous amino acids of the reference sequence, peptide 1; (b)an amino acid sequence substantially identical to the sequence definedin (a); and (c) a variant of the amino acid sequence defined in (a),which variant is selected from the group consisting of a substitutionvariant, a deletion variant, an addition variant, and combinationsthereof. In further embodiments, the peptides have an amino acidsequence having at least 9, at least 10, from 9 to 14, from 9 to 13,from 10 to 13, from 10 to 14, or the like contiguous amino acids of thereference sequence, peptide 1; an amino acid sequence substantiallyidentical thereto; or a variant thereof, which variant is selected fromthe group consisting of a substitution variant, a deletion variant, anaddition variant, and combinations thereof. As explained further below,one or more of the amino acids of the peptides (e.g., the N-terminaland/or C-terminal amino acids) may be optionally independentlychemically modified; in some embodiments, one or more amino acids of apeptide will be chemically modified while in other embodiments none ofthe amino acids of the peptide will be chemically modified. In oneaspect, preferred modification can occur at the amine (—NH₂) group ofthe N-terminal amino acid of the peptide or peptide segment (which aminegroup would form a peptide amide bond if present internally within apeptide sequence rather than at the N-terminal position). In anotheraspect, preferred modification can occur at the carboxy (—COOH) group ofthe C-terminal amino acid of the peptide or peptide segment (whichcarboxy group would form a peptide amide bond if present internallywithin a peptide sequence rather than at the C-terminal position). Inanother aspect, preferred modification can occur at both the N-terminalamine (—NH₂) group and at the C-terminal carboxylic (—COOH) group.

In some embodiments, the amino acid sequence of the peptide begins fromthe N-terminal amino acid of the reference sequence peptide 1. Forexample, the peptides may have an amino acid sequence selected from thegroup consisting of (a) an amino acid sequence having from 4 to 23contiguous amino acids of the reference sequence peptide 1, wherein theamino acid sequence begins from the N-terminal amino acid of thereference sequence (i.e., peptide 2, peptide 4, peptide 7, peptide 11,peptide 16, peptide 22, peptide 29, peptide 37, peptide 46, peptide 56,peptide 67, peptide 79, peptide 92, peptide 106, peptide 121, peptide137, peptide 154, peptide 172, peptide 191, or peptide 211); (b) asequence substantially similar to the amino acid sequence defined in(a); and (c) a variant of the amino acid sequence defined in (a). Thesepeptides contain no chemical moiety or a chemical moiety on theN-terminal glycine other than a myristoyl group. Preferably, thechemical moiety is an acyl group, in the form of an amide bond, such asan acetyl group, or alkyl group.

In other embodiments, the amino acid sequence of the peptide ends at theC-terminal amino acid of the reference sequence peptide 1. For example,the peptides may have an amino acid sequence selected from the groupconsisting of (a) an amino acid sequence having from 4 to 23 contiguousamino acids of the reference sequence peptide 1, wherein the amino acidsequence ends at the C-terminal amino acid of the reference sequence(i.e., peptide 3, peptide 6, peptide 10, peptide 15, peptide 21, peptide28, peptide 36, peptide 45, peptide 55, peptide 66, peptide 78, peptide91, peptide 105, peptide 120, peptide 136, peptide 153, peptide 171,peptide 190, peptide 210, or peptide 231); (b) a sequence substantiallysimilar to the amino acid sequence defined in (a); and (c) a variant ofthe amino acid sequence defined in (a).

In other embodiments, the amino acid sequence of the peptide does notbegin at the N-terminal amino acid of the reference sequence, peptide 1,(SEQ ID NO: 1) but rather begins at the amino acid at position 2 throughthe amino acid at position 21 of the reference sequence peptide 1. Forexample, the peptides may have an amino acid sequence selected from thegroup consisting of (a) an amino acid sequence having from 4 to 23contiguous amino acids of the reference sequence peptide 1, wherein theamino acid sequence begins at any amino acid between position 2 throughposition 21 of the reference sequence. These peptides may be between 4and 23 contiguous amino acids long and may represent peptides in themiddle of the reference sequence, peptide 1; (b) a sequencesubstantially similar to the amino acid sequence defined in (a); and (c)a variant of the amino acid sequence defined in (a). These peptides aredisclosed in Tables 1 or 2. These peptides may contain no covalentlybound chemical moiety or a chemical moiety on the N-terminal amino acidwhich is not the N-terminal glycine from or equivalent to the N-terminalglycine of the amino acid sequence SEQ ID NO: 1. Preferably, thechemical moiety is an acyl group, such as an acetyl group or a myristoylgroup, in the form of an amide bond, or an alkyl group.

Peptide amino acid sequences which are useful in the current inventionto inhibit mucin hypersecretion in a mammal, and which are useful toreduce the amount of mucin hypersecretion in a mammal, and which areuseful in the methods of inhibition of mucin hypersecretion and in themethods of reduction of mucin hypersecretion include amino acidsequences of isolated peptides and amino acid sequences of peptideswhich optionally contain N-terminal- and/or C-terminal-chemicallymodified groups of the current invention, which peptide amino acidsequences are selected from the group consisting of the 23-mers (i.e.,peptides having a 23 amino acid sequence): peptide 2; and peptide 3; the22-mers (i.e., peptides having a 22 amino acid sequence): peptide 4;peptide 5; and peptide 6; the 21-mers (i.e., peptides having a 21 aminoacid sequence): peptide 7; peptide 8; peptide 9; and peptide 10; the20-mers (i.e., peptides having a 20 amino acid sequence): peptide 11;peptide 12; peptide 13; peptide 14; and peptide 15; the 19-mers (i.e.,peptides having a 19 amino acid sequence): peptide 16; peptide 17;peptide 18; peptide 19; peptide 20; and peptide 21; the 18-mers (i.e.,peptides having a 18 amino acid sequence): peptide 22; peptide 23;peptide 25; peptide 26; peptide 27; and peptide 28; the 17-mers (i.e.,peptides having a 17 amino acid sequence): peptide 29; peptide 30;peptide 31; peptide 32; peptide 33; peptide 34; peptide 35; and peptide36; the 16-mers (i.e., peptides having a 16 amino acid sequence):peptide 37; peptide 38; peptide 39; peptide 40; peptide 41; peptide 42;peptide 43; peptide 44; and peptide 45; the 15-mers (i.e., peptideshaving a 15 amino acid sequence): peptide 46; peptide 47; peptide 48;peptide 49; peptide 50; peptide 51; peptide 52; peptide 53; peptide 54;and peptide 55; the 14-mers (i.e., peptides having a 14 amino acidsequence): peptide 56; peptide 57; peptide 58; peptide 59; peptide 60;peptide 61; peptide 62; peptide 63; peptide 64; peptide 65; and peptide66; the 13-mers (i.e., peptides having a 13 amino acid sequence):peptide 67; peptide 68; peptide 69; peptide 70; peptide 71; peptide 72;peptide 73; peptide 74; peptide 75; peptide 76; peptide 77; and peptide78; the 12-mers (i.e., peptides having a 12 amino acid sequence):peptide 79; peptide 80; peptide 81; peptide 82; peptide 83; peptide 84;peptide 85; peptide 86; peptide 87; peptide 88; peptide 89; peptide 90;and peptide 91; the 11-mers (i.e., peptides having a 11 amino acidsequence): peptide 92; peptide 93; peptide 94; peptide 95; peptide 96;peptide 97; peptide 98; peptide 99; peptide 100; peptide 101; peptide102; peptide 103; peptide 104; and peptide 105; the 10-mers (i.e.,peptides having a 10 amino acid sequence): peptide 106; peptide 107;peptide 108; peptide 109; peptide 110; peptide 111; peptide 112; peptide113; peptide 114; peptide 115; peptide 116; peptide 117; peptide 118;peptide 119; and peptide 120; the 9-mers (i.e., peptides having a 9amino acid sequence): peptide 121; peptide 122; peptide 123; peptide124; peptide 125; peptide 126; peptide 127; peptide 128; peptide 129;peptide 130; peptide 131; peptide 132; peptide 133; peptide 134; peptide135; and peptide 136; the 8-mers (i.e., peptides having a 8 amino acidsequence): peptide 137; peptide 138; peptide 139; peptide 140; peptide141; peptide 142; peptide 143; peptide 144; peptide 145; peptide 146;peptide 147; peptide 148; peptide 149; peptide 150; peptide 151; peptide152; and peptide 153; the 7-mers (i.e., peptides having a 7 amino acidsequence): peptide 154; peptide 155; peptide 156; peptide 157; peptide158; peptide 159; peptide 160; peptide 161; peptide 162; peptide 163;peptide 164; peptide 165; peptide 166; peptide 167; peptide 168; peptide169; peptide 170; and peptide 171; the 6-mers (i.e., peptides having a 6amino acid sequence): peptide 172; peptide 173; peptide 174; peptide175; peptide 176; peptide 177; peptide 178; peptide 179; peptide 180;peptide 181; peptide 182; peptide 183; peptide 184; peptide 185; peptide186; peptide 187; peptide 188; peptide 189; and peptide 190; the 5-mers(i.e., peptides having a 5 amino acid sequence): peptide 191; peptide192; peptide 193; peptide 194; peptide 195; peptide 196; peptide 197;peptide 198; peptide 199; peptide 200; peptide 201; peptide 202; peptide203; peptide 204; peptide 205; peptide 206; peptide 207; peptide 208;peptide 209; and peptide 210; and the 4-mers (i.e., peptides having a 4amino acid sequence): peptide 211; peptide 212; peptide 213; peptide214; peptide 215; peptide 216; peptide 217; peptide 218; peptide 219;peptide 220; peptide 221; peptide 222; peptide 223; peptide 224; peptide225; peptide 226; peptide 227; peptide 228; peptide 229; peptide 230;and peptide 231.

Preferred amino acid sequences of isolated peptides and of N-terminal-and/or C-terminal-chemically modified peptides of the current inventionare selected from the group consisting of the 23-mers: peptide 2; andpeptide 3; the 22-mers: peptide 4; peptide 5; and peptide 6; the21-mers: peptide 7; peptide 8; peptide 9; and peptide 10; the 20-mers:peptide 11; peptide 12; peptide 13; peptide 14; and peptide 15; the19-mers: peptide 16; peptide 17; peptide 18; peptide 19; peptide 20; andpeptide 21; the 18-mers: peptide 22; peptide 23; peptide 24; peptide 25;peptide 26; peptide 27; and peptide 28; the 17-mers: peptide 29; peptide30; peptide 31; peptide 32; peptide 33; peptide 34; peptide 35; andpeptide 36; the 16-mers: peptide 37; peptide 38; peptide 39; peptide 40;peptide 41; peptide 42; peptide 43; peptide 44; and peptide 45; the15-mers: peptide 46; peptide 47; peptide 48; peptide 49; peptide 50;peptide 51; peptide 52; peptide 53; and peptide 54; the 14-mers: peptide56; peptide 57; peptide 58; peptide 59; peptide 60; peptide 61; peptide62; peptide 63; and peptide 64; the 13-mers: peptide 67; peptide 68;peptide 69; peptide 70; peptide 71; peptide 72; peptide 73; peptide 74;and peptide 75; the 12-mers: peptide 79; peptide 80; peptide 81; peptide82; peptide 83; peptide 84; peptide 85; peptide 86; and peptide 87; the11-mers: peptide 92; peptide 93; peptide 94; peptide 95; peptide 96;peptide 97; peptide 98; peptide 99; and peptide 100; the 10-mers:peptide 106; peptide 107; peptide 108; peptide 109; peptide 110; peptide111; peptide 112; peptide 113; and peptide 114; the 9-mers: peptide 122;peptide 123; peptide 124; peptide 125; peptide 126; peptide 127; peptide128; and peptide 129; the 8-mers: peptide 139; peptide 140; peptide 141;peptide 142; peptide 143; peptide 144; and peptide 145; the 7-mers:peptide 157; peptide 158; peptide 159; peptide 160; peptide 161; andpeptide 162; the 6-mers: peptide 176; peptide 177; peptide 178; peptide179; and peptide 180; the 5-mers: peptide 196; peptide 197; peptide 198;and peptide 199; and the 4-mers: peptide 217; and peptide 219.

More preferred amino acid sequences of isolated peptides and ofN-terminal- and/or C-terminal-chemically modified peptides of thecurrent invention are selected from the group consisting of the 23-mers:peptide 2; and peptide 3; the 22-mers: peptide 4; peptide 5; and peptide6; the 21-mers: peptide 7; peptide 8; peptide 9; and peptide 10; the20-mers: peptide 11; peptide 12; peptide 13; peptide 14; and peptide 15;the 19-mers: peptide 16; peptide 17; peptide 18; peptide 19; peptide 20;and peptide 21; the 18-mers: peptide 22; peptide 23; peptide 24; peptide25; peptide 26; peptide 27; and peptide 28; the 17-mers: peptide 29;peptide 30; peptide 31; peptide 32; peptide 33; peptide 34; peptide 35;and peptide 36; the 16-mers: peptide 37; peptide 38; peptide 39; peptide40; peptide 41; peptide 42; peptide 43; peptide 44; and peptide 45; the15-mers: peptide 46; peptide 47; peptide 48; peptide 49; peptide 50;peptide 51; peptide 52; peptide 53; and peptide 54; the 14-mers: peptide56; peptide 57; peptide 58; peptide 59; peptide 60; peptide 61; peptide62; peptide 63; and peptide 64; the 13-mers: peptide 67; peptide 68;peptide 69; peptide 70; peptide 71; peptide 72; peptide 73; peptide 74;peptide 80; peptide 81; peptide 82; peptide 83; peptide 84; peptide 85;peptide 86; and peptide 87; the 11-mers: peptide 92; peptide 93; peptide94; peptide 95; peptide 96; peptide 97; peptide 98; peptide 99; andpeptide 100; the 10-mers: peptide 106; peptide 108; peptide 109; peptide110; peptide 111; peptide 112; peptide 113; and peptide 114; the 9-mers:peptide 124; peptide 125; peptide 126; peptide 127; peptide 128; andpeptide 129; the 8-mers: peptide 141; peptide 142; peptide 143; peptide144; and peptide 145; the 7-mers: peptide 159; peptide 160; peptide 161;and peptide 162; the 6-mers: peptide 178; peptide 179; and peptide 180;the 5-mers: peptide 198; and peptide 199; and the 4-mer: peptide 219.

In yet other embodiments, the amino acid sequence of the peptideincludes the contiguous residues A, K, G, and E as in peptide 219 of thereference sequence peptide 1. For example, the peptides may have anamino acid sequence selected from the group consisting of (a) an aminoacid sequence having from 4 to 23 contiguous amino acids of thereference sequence peptide 1, wherein the amino acid sequence of thepeptide includes the contiguous residues A, K, G, and E as in peptide219 of the reference peptide 1 (e.g., peptide 219, peptide 45, peptide79, peptide 67, peptide 80, etc.); (b) a sequence substantially similarto the amino acid sequence defined in (a); and (c) a variant of theamino acid sequence defined in (a).

Examples of peptide segments which contain the amino acid sequence AKGEof the reference peptide amino acid sequence, peptide 1, include (a) the23-mers: peptide 2; and peptide 3; the 22-mers: peptide 4; peptide 5;and peptide 6; the 11-mers: peptide 7; peptide 8; peptide 9; and peptide10; the 20-mers: peptide 11; peptide 12; peptide 13; peptide 14; andpeptide 15; the 19-mers: peptide 16; peptide 17; peptide 18; peptide 19;peptide 20; and peptide 21; the 18-mers: peptide 22; peptide 23; peptide24; peptide 25; peptide 26; peptide 27; and peptide 28; the 17-mers:peptide 29; peptide 30; peptide 31; peptide 32; peptide 33; peptide 34;peptide 35; and peptide 36; the 16-mers: peptide 37; peptide 38; peptide39; peptide 40; peptide 41; peptide 42; peptide 43; peptide 44; andpeptide 45; the 15-mers: peptide 46; peptide 47; peptide 48; peptide 49;peptide 50; peptide 51; peptide 52; peptide 53; and peptide 54; the14-mers: peptide 56; peptide 57; peptide 58; peptide 59; peptide 60;peptide 61; peptide 62; peptide 63; and peptide 64; the 13-mers: peptide67; peptide 68; peptide 69; peptide 70; peptide 71; peptide 72; peptide73; peptide 74; and peptide 75; the 12-mers: peptide 79; peptide 80;peptide 81; peptide 82; peptide 83; peptide 84; peptide 85; peptide 86;and peptide 87; the 11-mers: peptide 93; peptide 94; peptide 95; peptide96; peptide 97; peptide 98; peptide 99; and peptide 100; the 10-mers:peptide 108; peptide 109; peptide 110; peptide 111; peptide 112; peptide113; and peptide 114; the 9-mers: peptide 124; peptide 125; peptide 126;peptide 127; peptide 128; and peptide 129; the 8-mers: peptide 141;peptide 142; peptide 143; peptide 144; and peptide 145; the 7-mers:peptide 159; peptide 160; peptide 161; and peptide 162; the 6-mers:peptide 178; peptide 179; and peptide 180; the 5-mers: peptide 198; andpeptide 199; and the 4-mer: peptide 219, (b) a sequence substantiallysimilar to the amino acid sequence defined in (a); and (c) a variant ofthe amino acid sequence defined in (a), which variant is selected fromthe group consisting of a substitution variant, a deletion variant, anaddition variant, and combinations thereof, wherein the segmentcomprises or consists of from 4 to 23 contiguous amino acids.

In another embodiment, preferred peptide sequences have an amino acidsequence selected from the group consisting of (a) an amino acidsequence having from 10 to 23 contiguous amino acids of the referencesequence, peptide 1; (b) a sequence substantially similar to the aminoacid sequence defined in (a); and (c) a variant of the amino acidsequence defined in (a), which variant is selected from the groupconsisting of a substitution variant, a deletion variant, an additionvariant, and combinations thereof, wherein the preferred amino acidsequences comprise the 23-mer: peptide 2; the 22-mer: peptide 4; the21-mer: peptide 7; the 20-mer: peptide 11; the 19-mer: peptide 16; the18-mer: peptide 22; the 17-mer: peptide 29; the 16-mer: peptide 37; the15-mer: peptide 46; the 14-mer: peptide 56; the 13-mer: peptide 67; the12-mer: peptide 79; the 11-mer: peptide 92; and the 10-mer: peptide 106.

In further embodiments, the amino acid sequence of the peptide beginsfrom the N-terminal amino acid of the reference sequence peptide 1 andincludes the contiguous residues A, K, G, and E as in peptide 219 of thereference sequence peptide 1, while in other embodiments the amino acidsequence of the peptide ends at the C-terminal amino acid of thereference sequence peptide 1 and includes the contiguous residues A, K,G, and E as in peptide 219 of the reference sequence peptide 1.

The peptides may include one or more amino acid deletions,substitutions, and/or additions with respect to the reference amino acidsequence. Preferably, the substitutions may be conservative amino acidsubstitutions, or the substitutions may be non-conservative amino acidsubstitutions. In some embodiments, the peptides, including the peptideswith amino acid sequences that are substantially identical to orvariants of the reference amino acid sequence, will not have deletionsor additions as compared to the corresponding contiguous amino acids ofthe reference amino acid sequence, but may have conservative ornon-conservative substitutions. Amino acid substitutions that may bemade to the reference amino acid sequence in the peptides of theinvention include, but are not limited to, the following: alanine (A)may be substituted with lysine (K), valine (V), leucine (L), orisoleucine (I); glutamic acid (E) may be substituted with aspartic acid(D); glycine (G) may be substituted with proline (P); lysine (K) may besubstituted with arginine (R), glutamine (Q), or asparagine (N);phenylalanine (F) may be substituted with leucine (L), valine (V),isoleucine (I), or alanine (A); proline (P) may be substituted withglycine (G); glutamine (Q) may be substituted with glutamic acid (E) orasparagine (N); arginine (R) may be substituted with lysine (K),glutamine (Q), or asparagine (N); serine (S) may be substituted withthreonine; threonine (T) may be substituted with serine (S); and valine(V) may be substituted with leucine (L), isoleucine (I), methionine (M),phenylalanine (F), alanine (A), or norleucine (Nle). For example,substitutions that could be made to the reference amino acid sequence inthe peptides of the invention include substituting alanine (A) forphenylalanine (F) (e.g., at amino acid position 4 of the reference aminoacid sequence), glutamic acid (E) for glutamine (Q) (e.g., at amino acidposition 3 of the reference amino acid sequence), lysine (K) for alanine(A) (e.g., at amino acid positions 2 and/or 8 of the reference aminoacid sequence), and/or serine (S) for threonine (T) (e.g., at amino acidposition 7 of the reference amino acid sequence).

When substitutions are included in the amino acid sequences of thepeptides of the invention (which peptides comprise unmodified as well aspeptides which are chemically modified for example by N-terminal and/orC-terminal modification such as by amide formation) with respect to thereference amino acid sequence, there is preferably at least 80% sequenceidentity between the amino acid sequence of the peptide and thereference amino acid sequence. Peptides having 5 to 23 amino acids andincluding one amino acid substitution with respect to the referenceamino acid sequence will have between about 80% to about 96% (i.e.,˜95.7%) sequence identity to the reference amino acid sequence. Peptideshaving 10 to 23 amino acids and including one amino acid substitutionwith respect to the reference amino acid sequence will have betweenabout 90% to about 96% (i.e., ˜95.7%) sequence identity to the referenceamino acid sequence. Peptides having 20 to 23 amino acids and includingone amino acid substitution with respect to the reference amino acidsequence will have between about 95% to about 96% (i.e., ˜95.7%)sequence identity to the reference amino acid sequence. Peptides having10 to 23 amino acids and including two amino acid substitutions withrespect to the reference amino acid sequence will have between about 80%to about 92% (i.e., ˜91.3%) sequence identity to the reference aminoacid sequence. Peptides having 16 to 23 amino acids and including twoamino acid substitutions with respect to the reference amino acidsequence will have between about 87.5% to about 92% (i.e., ˜91.3%)sequence identity to the reference amino acid sequence. Peptides having20 to 23 amino acids and including two amino acid substitutions withrespect to the reference amino acid sequence will have between about 90%to about 92% (i.e., ˜91.3%) sequence identity to the reference aminoacid sequence. Peptides having 15 to 23 amino acids and including threeamino acid substitutions with respect to the reference amino acidsequence will have between about 80% to about 87% sequence identity tothe reference amino acid sequence. Peptides having 20 to 23 amino acidsand including three amino acid substitutions with respect to thereference amino acid sequence will have between about 85% to about 87%sequence identity to the reference amino acid sequence. Peptides having20 to 23 amino acids and including four amino acid substitutions withrespect to the reference amino acid sequence will have between about 80%to about 83% (i.e., ˜82.6%) sequence identity to the reference aminoacid sequence.

In peptides of the current invention, with respect to the contiguousamino acid sequence of the reference peptide (which is a 24-mer)substitution of one amino acid in a contiguous 23 amino acid sequence (a23-mer) selected from the reference 24 amino acid sequence provides apeptide with an amino acid sequence which has a 95.65% (or ˜96%)sequence identity to the amino acid segment in the reference peptidewith which the 23-mer has identity. Analogously, substitution of two,three, four, and five amino acids in said 23-mer provides a peptide withan amino acid sequence which has a 91.30% (or ˜91%), 86.96% (or ˜87%),82.61% (or ˜83%), and 78.27% (or ˜78%) sequence identity, respectively,to the reference peptide amino acid sequence. Analogously, substitutionof one, two, three, four, and five amino acids in a 22-mer provides apeptide with an amino acid sequence which has a 95.45% (or ˜95%), 90.91%(or ˜91%), 86.36% (or ˜86%), 81.82% (or ˜82%), and 77.27% (or ˜77%)sequence identity, respectively, to the reference peptide amino acidsequence. Analogously, substitution of one, two, three, four, and fiveamino acids in a 21-mer provides a peptide with an amino acid sequencewhich has a 95.24% (˜95%), 90.48 (˜91%), 85.71% (˜86%), 80.95 (˜81%),and 76.19% (˜76%) sequence identity, respectively, to the referencepeptide amino acid sequence. Analogously, substitution of one, two,three, four, and five amino acids in a 20-mer provides a peptide with anamino acid sequence which has a 95.00% (95%), 90.00% (90%), 85.00%(85%), 80.00% (80%), and 75.00% (75%) sequence identity, respectively,to the reference peptide amino acid sequence. Analogously, substitutionof one, two, three, and four amino acids in a 19-mer provides a peptidewith an amino acid sequence which has a 94.74% (˜95%), 89.47% (˜89%),84.21% (˜84%), and 78.95% (˜79%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of one,two, three, and four amino acids in an 18-mer provides a peptide with anamino acid sequence which has a 94.44% (˜94%), 88.89% (˜89%), 83.33%(˜83%), and 77.78% (˜78%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of one,two, three, and four amino acids in an 17-mer provides a peptide with anamino acid sequence which has a 94.12% (˜94%), 88.23% (˜88%), 82.35%(˜82%), and 76.47% (˜76%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of one,two, three, and four amino acids in a 16-mer provides a peptide with anamino acid sequence which has a 93.75% (˜94%), 87.50% (˜88%), 81.25%(˜81%), and 75.00% (75%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of one,two, and three amino acids in a 15-mer provides a peptide with an aminoacid sequence which has a 93.33% (˜93%), 86.67% (˜87%), and 80.00% (80%)sequence identity, respectively, to the reference peptide amino acidsequence. Analogously, substitution of one, two, and three amino acidsin a 14-mer provides a peptide with an amino acid sequence which has a92.86% (˜93%), 85.71% (˜86%), and 78.57% (79%) sequence identity,respectively, to the reference peptide amino acid sequence. Analogously,substitution of one, two, and three amino acids in a 13-mer provides apeptide with an amino acid sequence which has a 92.31% (˜92%), 84.62%(˜85%), and 76.92% (˜77%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of one,two, and three amino acids in a 12-mer provides a peptide with an aminoacid sequence which has a 91.67% (˜92%), 83.33% (˜83%), and 75.00% (75%)sequence identity, respectively, to the reference peptide amino acidsequence. Analogously, substitution of one and two amino acids in an11-mer provides a peptide with an amino acid sequence which has a 90.91%(˜91%) and 81.82% (˜82%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of oneand two amino acids in a 10-mer provides a peptide with an amino acidsequence which has a 90.00% (90%) and 80.00% (80%) sequence identity,respectively, to the reference peptide amino acid sequence. Analogously,substitution of one and two amino acids in a 9-mer provides a peptidewith an amino acid sequence which has a 88.89% (˜89%) and 77.78% (˜78%)sequence identity, respectively, to the reference peptide amino acidsequence. Analogously, substitution of one and two amino acids in an8-mer provides a peptide with an amino acid sequence which has a 87.50%(˜88%) and 75.00% (75%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of oneamino acid in a 7-mer, 6-mer, 5-mer, and 4-mer provides a peptide withan amino acid sequence which has a 85.71% (˜86%), 83.33% (˜83.3%),80.00% (80%), and 75.00% (75%) sequence identity, respectively, to thereference peptide. Preferred amino acid sequences of this invention havegreater than 80% sequence identity to the amino acid sequence in thereference sequence, more preferably between 81% and 96% sequenceidentity to the amino acid sequence in the reference sequence, and morepreferably between 80% and 96% sequence identity to the amino acidsequence in the reference sequence. The preferred amino acid sequencescan be optionally N-terminally chemically bonded at the terminal peptideamino group to a C2 to C22 linear aliphatic carboxylic acid moiety, morepreferably to a C2 to C16 linear aliphatic carboxylic acid moiety, mostpreferably to a C2 or C16 linear aliphatic carboxylic acid moiety, by anamide bond, and optionally C-terminally chemically bonded at theterminal peptide carboxylic group to an amine such as ammonia or aprimary or secondary amine such as a C1 to C16 linear aliphatic primaryamine, by an amide bond.

Examples of substitution variants of peptide 79, a 12-mer, include, forexample, peptide 238, where Q at position 3 in peptide 79 has beensubstituted by E in sequence 238; peptide 233, where A at position 2 inpeptide 79 has been substituted by K in peptide 233; peptide 234, whereA at position 8 in peptide 79 has been substituted by K in peptide 234;peptide 235, where A at positions 2 and 8 in peptide 79 have beensubstituted by K in peptide 235; peptide 237, where F at position 4 inpeptide 79 has been substituted by A in peptide 237; peptide 239, whereK at position 10 in peptide 79 has been substituted by A in peptide 239;peptide 240, where G at position 11 in peptide 79 has been substitutedby A in peptide 240; and peptide 241, where E at position 12 in peptide79 has been substituted by A in peptide 241.

Examples of substitution variants of peptide 106, a 10-mer, include, forexample, peptide 236, where F at position 4 in peptide 106 has beensubstituted by A in peptide 236; peptide 242, where G at position 1 inpeptide 106 has been substituted by A in peptide 242; peptide 243, whereQ at position 3 in peptide 106 has been substituted by A in peptide 243;peptide 244, where S at position 5 in peptide 106 has been substitutedby A in peptide 244; peptide 245, where K at position 6 in peptide 106has been substituted by A in peptide 245; peptide 247, where T atposition 7 in peptide 106 has been substituted by A in peptide 247;peptide 248, where K at position 10 in peptide 106 has been substitutedby A in peptide 248; peptide 249, where K at positions 6 and 10 inpeptide 106 have both been substituted, each by A, in peptide 249.

Examples of a substitution variant of peptide 137, an 8-mer, include forexample, peptide 250, where F at position 4 in peptide 137 has beensubstituted by A in peptide 250.

Examples of a substitution variant of peptide 219, a 4-mer, include forexample, peptide 251, where K at position 2 in peptide 219 has beensubstituted by A in peptide 251.

A substitution variant peptide such as described herein can be in theform of an isolated peptide or in the form of a chemically modifiedpeptide such as, for example, an N-terminal amide such as a myristoylamide, an acetyl amide, and the like as described herein, and such as,for example, a C-terminal amide such as an amide formed with ammonia,and such as both an N-terminal amide and a C-terminal amide.

When deletions are included in the amino acid sequences of the peptidesof the invention with respect to the reference amino acid sequence,there is preferably at least 80% sequence identity between the aminoacid sequence of the peptide to the reference amino acid sequence.Peptides having 5 to 23 amino acids and including one amino aciddeletion with respect to the reference peptide will have between 80% toabout 96% (i.e., ˜95.7%) sequence identity to the reference amino acidsequence. Peptides having 10 to 23 amino acids and including one aminoacid deletion with respect to the reference peptide will have betweenabout 90% to about 96% (i.e., ˜95.7%) sequence identity to the referenceamino acid sequence. Peptides having 20 to 23 amino acids and includingone amino acid deletion with respect to the reference peptide will havebetween 95% to about 96% (i.e., ˜95.7%) sequence identity to thereference amino acid sequence. Peptides having 10 to 23 amino acids andincluding two amino acid deletions with respect to the reference peptidewill have between about 80% to about 92% (i.e., ˜91.3%) sequenceidentity to the reference amino acid sequence. Peptides having 16 to 23amino acids and including two amino acid deletions with respect to thereference peptide will have between about 87.5% to about 92% (i.e.,˜91.3%) sequence identity to the reference amino acid sequence. Peptideshaving 20 to 23 amino acids and including two amino acid deletions withrespect to the reference peptide will have between about 90% to about92% (i.e., ˜91.3%) sequence identity to the reference amino acidsequence. Peptides having 15 to 23 amino acids and including three aminoacid deletions with respect to the reference peptide will have betweenabout 80% to about 87% sequence identity to the reference amino acidsequence. Peptides having 20 to 23 amino acids and including three aminoacid deletions with respect to the reference peptide will have betweenabout 85% to about 87% sequence identity to the reference amino acidsequence. Peptides having 20 to 23 amino and including four amino aciddeletions with respect to the reference peptide will have between about80% to about 83% (i.e., ˜82.6%) sequence identity to the reference aminoacid sequence.

As stated above, one or more of the amino acids of the peptides may alsobe chemically modified. Any amino acid modifications known in the artmay be made to the amino acids of the peptides using any method known inthe art.

In some embodiments, the N-terminal and/or C-terminal amino acid may bemodified. For example, the alpha-N-terminal amino acid of the peptidesmay be alkylated, amidated, or acylated at the alpha-N-terminal(N-terminal) amino (alpha-H2N—) group, and, for example, the C-terminalamino acid of the peptides may be amidated or esterified at theC-terminal carboxyl (—COOH) group. For example, the N-terminal aminogroup may be modified by acylation to include any acyl or fatty acylgroup to form an amide, including an acetyl group (i.e., CH3-C(═O)— or amyristoyl group, both of which are currently preferred groups). In someembodiments, the N-terminal amino group may be modified to include anacyl group having formula —C(O)R, wherein R is a linear or branchedalkyl group having from 1 to 15 carbon atoms, or may be modified toinclude an acyl group having formula —C(O)R1, wherein R1 is a linearalkyl group having from 1 to 15 carbon atoms. The N-amide can also be aformamide (R═H). The C-terminal amino acid of the peptides may also bechemically modified. For example, the C-terminal carboxyl group of theC-terminal amino acid may be chemically modified by conversion to acarboxamide group in place of the carboxyl group. (i.e., amidated). Insome embodiments, the N-terminal and/or C-terminal amino acids are notchemically modified. In some embodiments, the N-terminal group ismodified and the C-terminal group is not modified. In some embodiments,both the N-terminal and the C-terminal groups are modified.

The peptide may be acylated at the amino group of the N-terminal aminoacid to form an N-terminal amide with an acid selected from the groupconsisting of:

(i-a) a C2 (acetyl) to C13 aliphatic (saturated or optionallyunsaturated) carboxylic acid (for example, an N-terminal amide withacetic acid (which is a preferred group), with propanoic acid, withbutanoic acid, with hexanoic acid, with octanoic acid, with decanoicacid, with dodecanoic acid) which may be linear, branched (greater thanC3), or comprise a ring (greater than C3);

(i-b) a saturated C14 aliphatic carboxylic acid, which may be linear,branched or comprise a ring;

(i-c) an unsaturated C14 aliphatic carboxylic acid, which may be linear,branched or comprise a ring;

(i-d) C15 to C24 aliphatic (saturated or optionally unsaturated)carboxylic acid, which may be linear, branched or comprise a ring (forexample, with tetradecanoic acid (myristic acid which is a preferredgroup), with hexadecanoic acid, with 9-hexadecenoic acid, withoctadecanoic acid, with 9-octadecenoic acid, with 11-octadecenoic acid,with 9,12-octadecadienoic acid, with 9,12,15-octadecatrienoic acid, with6,9,12-octadecatrienoic acid, with eicosanoic acid, with 9-eicosenoicacid, with 5,8,11,14-eicosatetraenoic acid, with5,8,11,14,17-eicosapentaenoic acid, with docosanoic acid, with13-docosenoic acid, with 4,7,10,13,16,19-docosahexaenoic acid, withtetracosanoic acid, and the like);

(ii) trifluoroacetic acid;

(iii) benzoic acid; and

(iv-a) a C1 to C12 aliphatic alkyl sulfonic acid which forms analiphatic alkyl sulfonamide, wherein the C1 to C12 aliphatic alkylcarbon chain structure of the sulfonic acid is analogous to that of thealiphatic alkyl carboxylic acid chains in the aliphatic alkyl carboxylicacids described above. For example, a peptide may be acylated using acarboxylic acid group represented as (C1-C11)-alkyl-C(O)OH throughdehydrative coupling by way of activation of the carboxylic acid groupto form an amide represented as (C1-C11-alkyl-C(O)—NH-peptide.Analogously, a sulfonamide may be formed by reacting a sulfonic acidspecies (represented as (C1-C12)-alkyl-S(O2)-X, e.g., where X is halogenor OCH3 or other compatible leaving group) with an N-terminal aminogroup to form a sulfonamide represented as(C1-C12)-alkyl-S(O2)-NH-peptide.

(iv-b) a C14 to C24 aliphatic alkyl sulfonic acid which forms analiphatic alkyl sulfonamide, wherein the C14 to C24 aliphatic alkylcarbon chain structure of the sulfonic acid is analogous to that of thealiphatic alkyl carboxylic acid chains in the aliphatic alkyl carboxylicacids described above . . . . For example, a peptide may be acylatedusing a carboxylic acid group represented as (C13-C23)-alkyl-C(O)OHthrough dehydrative coupling by way of activation of the carboxylic acidgroup to form an amide represented as (C13-C23)-alkyl-C(O)—NH-peptide.Analogously, a sulfonamide may be formed by reacting a sulfonic acidspecies (represented as (C14-C24)-alkyl-S(O2)-X, e.g., where X ishalogen or OCH3 or other compatible leaving group) with an N-terminalamino group to form a sulfonamide represented as(C14-C24)-alkyl-S(O2)-NH-peptide.

As another example, the N-terminal amino group of the N-terminal aminoacid may be alkylated with a C1 to C12 aliphatic alkyl group, thestructure of which aliphatic alkyl group is as described above.Alkylation may be effected, for example, using an aliphatic alkyl halideor an aliphatic alkyl sulfonic acid ester (mesylate, tosylate, etc.),preferably using a primary alkyl halide or a primary alkyl sulfonic acidester. The N-terminal amino acid may be also modified at the terminalamino to include any acyl or aliphatic acyl fatty acyl group as anamide, including an acetyl group (i.e., —C(O)CH3, which is a preferredgroup), a myristoyl group (which is a preferred group), a butanoylgroup, a hexanoyl group, a octanoyl group, a decanoyl group, adodecanoyl group, a tetradecanoyl group, a hexadecanoyl group, a9-hexadecenoyl group, a octadecanoyl group, a 9-octadecenoyl group, a11-octadecenoyl group, a 9,12-octadecadienoyl group, a9,12,15-octadecatrienoyl group, a 6,9,12-octadecatrienoyl group, aeicosanoyl group, a 9-eicosenoyl group, a 5,8,11,14-eicosatetraenoylgroup, a 5,8,11,14,17-eicosapentaenoyl group, a docosanoyl group, a13-docosenoyl group, a 4,7,10,13,16,19-docosahexaenoyl group, atetracosanoyl group, which groups are covalently attached to theterminal amino group of the peptide by an amide bond.

The C-terminal carboxylic acid group of the C-terminal amino acid of thepeptides of the invention may also be chemically modified. For example,the C-terminal amino acid may be chemically modified by reaction of theC-terminal carboxylic acid group of the peptide with an amine to form anamide group such as an amide of ammonia which is a preferred group; anamide of a C1 to C12 aliphatic alkyl amine, preferably a linearaliphatic alkyl amine; an amide of a hydroxyl-substituted C2 to C12aliphatic alkyl amine; an amide of a linear 2-(C1 to C12 aliphaticalkyl)oxyethylamine group; and an amide of anomega-methoxy-poly(ethyleneoxy)n-ethylamine group (also referred to asan omega-methoxy-PEG-alpha-amine group or an omega-methoxy-(polyethyleneglycol)amine group), where n is from 0 to 10. The C-terminal carboxylicacid group of the C-terminal amino acid of the peptide may also be inthe form of an ester selected from the group consisting of an ester of aC1 to C12 aliphatic alkyl alcohol and an ester of a2-(omega-methoxy-poly(ethyleneoxy)n)-ethanol (MPEG) group, where n isfrom 0 to 10. In one aspect, a polyethylene glycol component such as ina PEG ester, an MPEG ester, a PEG amide, an MPEG amide and the likepreferably has a molecular weight of from about 500 to 40,000 Daltons,more preferably from 1000 to 25,000 Daltons, and most preferably fromabout 1000 to about 10,000 Daltons.

The C-terminal carboxylic acid group on the peptide, which may berepresented by the formula peptide-C(O)OH, may also be amidated byconversion to an activated form such as a carboxylic acid halide,carboxylic acid anhydride, N-hydroxysuccinimide ester, pentafluorophenyl(OPfp) ester, 3-hydroxy-2,3-dihydro-4-oxo-benzo-triazone (ODhbt) ester,and the like to facilitate reaction with ammonia or a primary orsecondary amine, preferably ammonia or a primary amine, and preferablywhile any other reactive groups in the peptide are protected bysynthetic chemically compatible protecting groups well known in the artof peptide synthesis, especially of peptide solid phase synthesis, suchas a benzyl ester, a t-butyl ester, a phenyl ester, etc. A resultingpeptide amide could be represented by the formula peptide-C(O)—NR3R4(the amide being at the C-terminal end of the peptide) wherein R3 and R4are independently selected from the group consisting of hydrogen; C1 toC12 alkyl such as methyl, ethyl, butyl, isobutyl, cyclopropylmethyl,hexyl, dodecyl, and optionally higher e.g., from C14 to C24 such astetradecyl, and the like as described above.

The C-terminal carboxylic acid of the C-terminal amino acid may also beconverted to an amide of a hydroxyl-substituted C2 to C12 aliphaticalkyl amine (the hydroxyl group being attached to a carbon atom ratherthan a nitrogen atom of the amine) such as 2-hydroxyethylamine,4-hydroxybutylamine, and 12-hydroxydodecylamine, and the like.

The C-terminal carboxylic acid may also be converted to an amide of ahydroxyl-substituted C2 to C12 aliphatic alkyl amine, wherein thehydroxyl group can be acylated to form an ester with a C2 to C12aliphatic carboxylic acid as described above. Preferably, in the peptideamide at the C-terminal end of the peptide represented by the formulapeptide-C(O)NR5R6, R5 is hydrogen and R6 is selected from the groupconsisting of hydrogen, C1 to C12 alkyl, and hydroxyl-substituted C2 toC12 alkyl.

The C-terminal carboxylic acid of the C-terminal amino acid may beconverted to an amide of a linear 2-(C1 to C12 aliphaticalkyl)oxyethylamine. Such an amide may be prepared, for example, byreaction of a linear C1 to C12 aliphatic alcohol with potassium hydridein diglyme with 2-chloroethanol to provide a linear C1 to C12 aliphaticalkyl ethanol, which can be converted to an amine by oxidation to analdehyde, followed by reductive amination to an amine (for example usingammonia), or by conversion to an alkyl halide (e.g. using thionylchloride) followed by treatment with an amine such as ammonia.

The C-terminal carboxylic acid of the C-terminal amino acid may beconverted to an amide of a linear PEG-amine (e.g.,omega-hydroxy-PEG-alpha-amine; omega-(C1-to-C12)-PEG-alpha-amine such asomega-methoxy-PEG-alpha-amine, i.e., MPEG-amine). In one aspect, thepolyethylene glycol or PEG component preferably has a molecular weightof from about 500 to 40,000 Daltons, more preferably from 1000 to 25,000Daltons, and most preferably from about 1000 to about 10,000 Daltons.

The C-terminal carboxylic acid of the C-terminal amino acid may also beconverted to an amide of an omega-methoxy-poly(ethyleneoxy)n-ethylamine,where n is from 0 to 10, which can be prepared from the correspondingomega-methoxy-poly(ethyleneoxy)n-ethanol, for example, by conversion ofthe alcohol to an amine as described above.

In another embodiment, the C-terminal carboxyl may be converted to anamide represented by the formula peptide-C(O)—NR7R8, wherein R7 ishydrogen and R8 is a linear 2-(C1 to C12 aliphatic alkyl)oxyethyl groupwherein the C1 to C12 aliphatic alkyl portion is as described above andincludes groups such as methoxyethyl (i.e., CH3O—CH2CH2-),2-dodecyloxyethyl, and the like; or R7 is hydrogen and R8 is anomega-methoxy-poly(ethyleneoxy)n-ethyl group where the n of thepoly(ethyleneoxy) portion is from 0 to 10, such as 2-methoxyethyl (i.e.,CH3O—CH2CH2-), omega-methoxyethoxyethyl (i.e., CH3O—CH2CH2O—CH2CH2-) upto CH3O—(CH2CH2O)10-CH2CH2-.

The C-terminal carboxylic acid group of the C-terminal amino acid of thepeptide may also be in the form of an ester of a C1 to C12 aliphaticalkyl alcohol, the aliphatic alkyl portion of the alcohol as describedabove. The C-terminal carboxylic acid group of the C-terminal amino acidof the peptide may also be in the form of an ester of a2-(omega-methoxy-poly(ethyleneoxy)n)-ethanol group where n is from 0 to10, which can be prepared from reaction of 2-methoxyethanol as a sodium2-methoxyethanolate with stoichiometric amounts of ethylene oxide, thestoichiometric amount dependent on the size of n.

A side chain in an amino acid of the peptides may also be chemicallymodified. For example, a phenyl group in phenylalanine or tyrosine maybe substituted with a substituent selected from the group consisting of:

a C1 to C24 aliphatic alkyl group (i.e., linear or branched, and/orsaturated or unsaturated, and/or containing a cyclic group) such asmethyl (preferred), ethyl, propyl, isopropyl, butyl, isobutyl,cyclopropyl, 2-methylcyclopropyl, cyclohexyl, octyl, decyl, dodecyl,hexadecyl, octadecyl, eicosanyl, docosanyl, tetracosanyl, 9-hexadecenyl,9-octadecenyl, 11-octadecenyl, 9,12-octadecadienyl,9,12,15-octadecatrienyl, 6,9,12-octadecatrienyl, 9-eicosenyl,5,8,11,14-eicosatetraenyl, 5,8,11,14,17-eicosapentaenyl, 13-docosenyl,and 4,7,10,13,16,19-docosahexaenyl;

a C1 to C12 aliphatic alkyl group substituted with a hydroxyl group atleast one carbon atom away from a site of unsaturation, examples ofwhich hydroxyalkyl group include hydroxymethyl, hydroxyethyl,hydroxydodecyl, and the like;

a C1 to C12 alkyl group substituted with a hydroxyl group that isesterified with a C2 to C25 aliphatic carboxyl group of an acid such asacetic acid, butanoic acid, hexanoic acid, octanoic acid, decanoic acid,dodecanoic acid, tetradecanoic acid, hexadecanoic acid, 9-hexadecenoicacid, octadecanoic acid, 9-octadecenoic acid, 11-octadecenoic acid,9,12-octadecadienoic acid, 9,12,15-octadecatrienoic acid,6,9,12-octadecatrienoic acid, eicosanoic acid, 9-eicosenoic acid,5,8,11,14-eicosatetraenoic acid, 5,8,11,14,17-eicosapentaenoic acid,docosanoic acid, 13-docosenoic acid, 4,7,10,13,16,19-docosahexaenoicacid, tetracosanoic acid, and the like, a dicarboxylic acid such assuccinic acid, or a hydroxyacid such as lactic acid, wherein the totalnumber of carbon atoms of the ester substituent is between 3 and 25;

halogen such as fluoro-, chloro-, bromo-, and iodo-; nitro-;

amino—such as NH2, methyl amino, dimethylamino; trifluoromethyl-;

carboxyl (—COOH);

a C1 to C24 alkoxy (such as can be formed by alkylation of tyrosine)such as methoxy, ethoxy, propyloxy, isopropyloxy, butyloxy, isobutyloxy,cyclopropyloxy, 2-methoxycyclopropyloxy, cyclohexyloxy, octyloxy,decyloxy, dodecyloxy, hexadecyloxy, octadecyloxy, eicosanyloxy,docosanyloxy, tetracosanyloxy, 9-hexadecenyloxy, 9-octadecenyloxy,11-octadecenyloxy, 9,12-octadecadienyloxy, 9,12,15-octadecatrienyloxy,6,9,12-octadecatrienyloxy, 9-eicosenyloxy, 5,8,11,14-eicosatetraenyloxy,5,8,11,14,17-eicosapentaenyloxy, 13-docosenyloxy, and4,7,10,13,16,19-docosahexaenyloxy; and a C2 to C12 hydroxyalkyloxy suchas 2-hydroxyethyloxy and esters thereof with carboxylic acids asdescribed above or with trifluoroacetic acid.

A serine hydroxyl group may be esterified with a substituent selectedfrom the group consisting of:

a C2 to C12 aliphatic carboxylic acid group such as described above;

a trifluoroacetic acid group; and

a benzoic acid group.

The epsilon amino group in lysine may be chemically modified, forexample, by amide formation with: a C2 to C12 aliphatic carboxylic acidgroup (for example, by reaction of the amine with a chemically activatedform of a carboxylic acid such as an acid chloride, an anhydride, anN-hydroxysuccinimide ester, a pentafluorophenyl (OPfp) ester, a3-hydroxy-2,3-dihydro-4-oxo-benzo-triazone (ODhbt) ester, and the like)such as described above, or a benzoic acid group, or an amino acidgroup. Additionally, the epsilon amino group in lysine may be chemicallymodified by alkylation with one or two C1 to C4 aliphatic alkyl groups.

The carboxylic acid group in glutamic acid may be modified by formationof an amide with an amine such as: ammonia; a C1 to C12 primaryaliphatic alkyl amine (the alkyl portion of which is as described above)including with methylamine; or an amino group of an amino acid.

The carboxylic acid group in glutamic acid may be modified by formationof an ester with a C1 to C12 aliphatic hydroxyalkyl group as describedabove, preferably an ester with a primary alcohol of a C1 to C12aliphatic alkyl such as methanol, ethanol, propan-1-ol, n-dodecanol, andthe like as described above.

In a preferred embodiment, the present invention comprises a method ofinhibiting the release of at least one inflammatory mediator from agranule in at least one inflammatory cell in a tissue and/or fluid of asubject comprising administration to said tissue and/or fluid atherapeutically effective amount of a pharmaceutical compositioncomprising at least one peptide having an amino acid sequence selectedfrom the group consisting of:

(a) an amino acid sequence having from 4 to 23 contiguous amino acids ofa reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1);

(b) an amino acid sequence having the sequence, GAQFSKTAAKGEAAAERPGEAAVA(SEQ ID NO. 1); and

(c) an amino acid sequence substantially identical to the sequencedefined in (a), wherein the C-terminal amino acid of the peptide isoptionally independently chemically modified, and the N-terminal aminoacid of the peptide is independently chemically modified by acylationwith a carboxylic acid selected from the group consisting of a C2 to C13saturated or unsaturated aliphatic carboxylic acid, a C14 saturated(myristic acid) or unsaturated aliphatic carboxylic acid, a C15 to C24saturated or unsaturated aliphatic carboxylic acid, and trifluoroaceticacid, or is not chemically modified, with the proviso that said peptidecan be modified by acylation when its amino acid sequence begins withthe sequence GAQF of the reference sequence by acylation only with acarboxylic acid selected from the group consisting of a C2 to C13saturated or unsaturated aliphatic carboxylic acid, a C14 unsaturatedaliphatic carboxylic acid, a C15 to C24 saturated or unsaturatedaliphatic carboxylic acid, and trifluoroacetic acid, or is notchemically modified, wherein said peptide, optionally combined with apharmaceutically acceptable carrier, and in a therapeutically effectiveinflammatory mediator release-reducing amount to reduce the release ofsaid inflammatory mediator from at least one inflammatory cell ascompared to release of said inflammatory mediator from at least one ofthe same type of inflammatory cell that would occur in the absence ofsaid at least one peptide.

The method preferably employs a peptide that can be acetylated at thealpha N-terminal amino acid. This peptide can consist of at least tencontiguous amino acid residues and is preferably embodied byacetyl-peptide 106 (SEQ ID NO: 106).

The method also employs a peptide consisting of at least four contiguousamino acid residues and more preferably at least six contiguous aminoacid residues. Further, the peptide can be myristoylated at the alphaN-terminal amino acid when the peptide. The method also can utilizedpeptide that can be amidated with ammonia at the alpha C-terminal aminoacid.

The method in a further embodiment utilizes a peptide comprises an aminoacid sequence of (a) an amino acid sequence having from 4 to 23contiguous amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA(SEQ ID NO. 1), wherein the N-terminal amino acid of the amino acidsequence of (a) is selected from amino acid position 2 to 21 of thereference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1). Further,these peptides can be myristoylated at the alpha N-terminal amino acidand also can be amidated with ammonia at the alpha C-terminal aminoacid.

The method of administration according to the present invention definesthe reduction of the release of an inflammatory mediator as blocking orinhibiting the mechanism that releases an inflammatory mediator from theinflammatory cell in said subject.

The method of administration includes incorporating or mixing thedisclosed peptides with a pharmaceutically acceptable carrier to form apharmaceutical composition.

The method of administration of the present invention release of atleast one inflammatory mediator release-reducing amount to reduce therelease of said inflammatory mediator from at least one inflammatorycell as compared to release of said inflammatory mediator from at leastone of the same type of inflammatory cell that would occur in theabsence of said at least one peptide. The inflammatory cell in saidsubject can be a leukocyte, a granulocyte, a basophil, an eosinophil,monocyte, macrophage or a combination thereof.

The inflammatory mediator released from at least one granule of at leastone inflammatory cell is selected from the group consisting ofmyeloperoxidase (MPO), eosinophil peroxidase (EPO), major basic protein[MBP], lysozyme, granzyme, histamine, proteoglycan, protease, achemotactic factor, cytokine, a metabolite of arachidonic acid,defensin, bactericidal permeability-increasing protein (BPI), elastase,cathepsin G, cathepsin B, cathepsin D, beta-D-glucuronidase,alpha-mannosidase, phospholipase A₂, chondroitin-4-sulphate, proteinase3, lactoferrin, collagenase, complement activator, complement receptor,N-formylmethionyl-leucyl-phenylalanine (FMLP) receptor, lamininreceptor, cytochrome b₅₅₈, monocyte-chemotactic factor, histaminase,vitamin B12 binding protein, gelatinase, plasminogen activator,beta-D-glucuronidase, and a combination thereof. Preferably theinflammatory mediator is selected from the group consisting ofmyeloperoxidase (MPO), eosinophil peroxidase (EPO), major basic protein(MBP), lysozyme, granzyme and a combination thereof.

The method according to claim 13, wherein said effective inflammatorymediator release-reducing amount of said peptide comprises adegranulation-inhibiting amount of peptide that reduces the amount of aninflammatory mediator released from at least one inflammatory cell fromabout 1% to about 99% or preferably about 5-50% to about 99%, ascompared to the amount released from at least one inflammatory cell inthe absence of the peptide.

The method of the present invention is useful for the treatment of asubject afflicted by or suffering from a respiratory disease. Thisrespiratory disease may be asthma, chronic bronchitis, chronicobstructive pulmonary disease (COPD) and cystic fibrosis. The subjectsthat can be treated by the present invention are preferably mammals,such as humans, canines, equines and felines.

The method of administration of the peptides of the present inventionare by topical administration, parenteral administration, rectaladministration, pulmonary administration, nasal administration, and oraladministration. More preferably, the pulmonary administration comprisesan aerosol, which can be generated from a dry powder inhaler, a metereddose inhaler or nebulizer. Additionally, the administration to thesubject can further include the administration of a second moleculeselected from the group consisting of an antibiotic, an antiviralcompound, an antiparasitic compound, an anti-inflammatory compound, andan immunomodulator.

The method is also useful for the treatment of a subject who isafflicted by or suffering from a disease selected from the groupconsisting of a bowel disease, a skin disease, an autoimmune disease, apain syndrome, and combinations thereof. More specifically, the boweldisease is selected from the group consisting of ulcerative colitis,Crohn's disease and irritable bowel syndrome. Skin diseases alsotreatable by the present method includes rosacea, eczema, psoriasis andsevere acne. Additionally a subject suffering from arthritis may also betreated by the present invention.

The present invention in one embodiment encompasses the administrationof peptides comprising an amino acid sequence substantially identical tothe amino acid sequence of (a) having from 4 to 23 contiguous aminoacids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1).These peptides preferably are selected from the group consisting of SEQID NOS: 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,247, 248, 249, 250, 251 and 252. These peptides can be furtheracetylated at the alpha N-terminal amino acid or myristoylated at thealpha N-terminal amino acid and optionally amidated with ammonia at thealpha C-terminal amino acid.

The method of the present invention also is useful for reducing mucushypersecretion in a subject by the administration of the peptides of thepresent invention as described herein for also reducing MARCKS-relatedmucus hypersecretion from at least one mucus secreting cell or tissue inthe subject, whereby mucus hypersecretion in the subject is reducedcompared to that which would occur in the absence of said administrationof said peptide.

The present invention is directed to an isolated peptide having an aminoacid sequence selected from the group consisting of:

(a) an amino acid sequence having from 4 to 23 contiguous amino acids ofa reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1);

(b) an amino acid sequence having the sequence, GAQFSKTAAKGEAAAERPGEAAVA(SEQ ID NO. 1); and

(c) an amino acid sequence substantially identical to the sequencedefined in (a),

wherein the C-terminal amino acid of the peptide is optionallyindependently chemically modified, and the N-terminal amino acid of thepeptide is independently chemically modified by acylation with acarboxylic acid selected from the group consisting of a C2 to C13saturated or unsaturated aliphatic carboxylic acid, a C14 saturated orunsaturated aliphatic carboxylic acid, a C15 to C24 saturated orunsaturated aliphatic carboxylic acid, and trifluoroacetic acid, or isnot chemically modified, with the proviso that said peptide is modifiedby acylation when its amino acid sequence begins with the sequence GAQFof the reference sequence by acylation only with a carboxylic acidselected from the group consisting of a C2 to C13 saturated orunsaturated aliphatic carboxylic acid, a C14 unsaturated aliphaticcarboxylic acid, a C15 to C24 saturated or unsaturated aliphaticcarboxylic acid, and trifluoroacetic acid, or is not chemicallymodified, wherein said peptide, optionally combined with apharmaceutically acceptable carrier, and in a therapeutically effectiveinflammatory mediator release-reducing amount to reduce the release ofsaid inflammatory mediator from at least one inflammatory cell ascompared to release of said inflammatory mediator from at least one ofthe same type of inflammatory cell that would occur in the absence ofsaid at least one peptide.

The isolated peptide can be acetylated at the alpha N-terminal aminoacid. The isolated peptide consists of at least ten contiguous aminoacid residues and preferably is an isolated peptide consists ofacetyl-peptide 106 (SEQ ID NO: 106).

In a further embodiment, the peptide consists of at least fourcontiguous amino acid residues or peptide consists of at least sixcontiguous amino acid residues.

The peptide can also be myristoylated at the alpha N-terminal amino acidand/or peptide can be amidated with ammonia at the alpha C-terminalamino acid.

The isolated peptide can further comprise an amino acid sequence of (a)described above, (a) an amino acid sequence having from 4 to 23contiguous amino acids of a reference sequence, GAQFSKTAAKGEAAAERPGEAAVA(SEQ ID NO. 1); wherein the N-terminal amino acid of the amino acidsequence of (a) is selected from amino acid position 2 to 21 of thereference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1). Thispeptide can be further myristoylated or acetylated at the alphaN-terminal amino acid or optionally amidated with ammonia at the alphaC-terminal amino acid.

The isolated peptide in a further embodiment, wherein the amino acidsequence is substantially identical to the amino acid sequence of (a)having from 4 to 23 contiguous amino acids of a reference sequence,GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1). These peptides preferably areselected from the group consisting of SEQ ID NOS: 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248, 249, 250, 251 and252. These peptides can be further acetylated at the alpha N-terminalamino acid or myristoylated at the alpha N-terminal amino acid andoptionally amidated with ammonia at the alpha C-terminal amino acid.amino acid sequence of (c) substantially identical to the amino acidsequence of (a) is selected from the group consisting of SEQ ID NOS:233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247,248, 249, 250, 251 and 252.

The invention also encompasses a composition comprising an isolatedpeptide as described in the paragraphs above and described herein and anexcipient. The invention also encompasses a pharmaceutical compositioncomprising an isolated peptide an isolated peptide as described in theparagraphs above and described herein and a pharmaceutically acceptablecarrier. The pharmaceutical composition can further preferably besterile, sterilizable or sterilized. These peptides can be contained ina kit with reagents useful for administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate that PKC-dependent phosphorylation releasesMARCKS from the plasma membrane to the cytoplasm.

FIGS. 2A-2C show that PKG induces dephosphorylation of MARCKS byactivating PP2A.

FIG. 3 depicts bar graphs that demonstrate that PP2A is an essentialcomponent of the mucin secretory pathway.

FIG. 4 is a gel that illustrates that MARCKS associates with actin andmyosin in the cytoplasm.

FIG. 5 depicts a signaling mechanism controlling MPO secretion inneutrophils.

FIG. 6 is a bar graph depicting the ability of MANS peptide to blocksecretion of myloperoxidase from isolated canine neutrophils.

FIG. 7 is a bar graph depicting the ability of MANS peptide to blocksecretion of myloperoxidase from isolated human neutrophils.

FIG. 8 is a bar graph showing that PMA stimulates a small increase inMPO secretion from LPS-stimulated human neutrophils which is enhanced ina concentration-dependent manner by co-stimulation with 8-Br-cGMP.

FIG. 9 is a bar graph showing that 8-Br-cGMP simulation has littleeffect on MPO secretion from LPS-stimulated human neutrophils until aco-stimulation with PMA occurs in a concentration-dependent manner.

FIG. 10 is a bar graph showing that PMA stimulates a small increase inMPO secretion from LPS-stimulated canine neutrophils which is enhancedin a concentration-dependent manner by co-stimulation with 8-Br-cGMP.

FIG. 11 is a bar graph showing that 8-Br-cGMP simulation has littleeffect on MPO secretion from LPS-stimulated canine neutrophils until aco-stimulation with PMA occurs in a concentration-dependent manner.

FIG. 12 is a bar graph showing that co-stimulation with PMA+8-Br-cGMP isrequired for maximal MPO secretion from LPS-stimulated canineneutrophils.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter withreference to the accompanying figures, in which preferred embodiments ofthe invention are illustrated. This invention may, however, be embodiedin different forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. The use of the words “a” or“an” herein to describe any aspect of the present invention is to beinterpreted as indicating one or more.

The present invention is directed to a method of inhibiting theexocytotic release of at least one inflammatory mediator from at leastone inflammatory cell comprising contacting the at least oneinflammatory cell, which cell comprises at least one inflammatorymediator contained within a vesicle inside the cell, with at least onepeptide selected from the group consisting of a MANS peptide and anactive fragment thereof in an effective amount to reduce the release ofthe inflammatory mediator from the inflammatory cell as compared to therelease of the inflammatory mediator from the same type of inflammatorycell that would occur in the absence of the at least one peptide.

The present invention is further directed to a method of inhibiting therelease of at least one inflammatory mediator from at least oneinflammatory cell in a tissue or fluid of a subject comprising theadministration to the subject's tissue and/or fluid, which comprises atleast one inflammatory cell comprising at least one inflammatorymediator contained within a vesicle inside the cell, a therapeuticallyeffective amount of a pharmaceutical composition comprising at least onepeptide selected from the group consisting of a MANS peptide and anactive fragment thereof in a therapeutically effective amount to reducethe release of the inflammatory mediator from at least one inflammatorycell as compared to release of the inflammatory mediator from at leastone of the same type of inflammatory cell that would occur in theabsence of the at least one peptide. More specifically, reducing therelease of an inflammatory mediator comprises blocking or inhibiting themechanism that releases an inflammatory mediator from the inflammatorycell.

The present invention is directed to the contact and/or administrationof the peptide described above and throughout the specification with anyknown inflammatory cell that may be contained in the tissue or fluid ofa subject which contains at least one inflammatory mediator containedwithin a vesicle inside the cell. The inflammatory cell is preferably aleukocyte, more preferably a granulocyte, which can be furtherclassified as a neutrophil, a basophil, an eosinophil or a combinationthereof. The inflammatory cells contacted in the present method may alsobe a monocyte/macrophage.

The present invention is directed to reducing the release ofinflammatory mediators contained within the vesicles of inflammatorycells and these inflammatory mediators are selected from the groupconsisting of myeloperoxidase (MPO), eosinophil peroxidase (EPO), majorbasic protein (MBP), lysozyme, granzyme, histamine, proteoglycan,protease, a chemotactic factor, cytokine, a metabolite of arachidonicacid, defensin, bactericidal permeability-increasing protein (BPI),elastase, cathepsin G, cathepsin B, cathepsin D, beta-D-glucuronidase,alpha-mannosidase, phospholipase A₂, chondroitin-4-sulphate, proteinase3, lactoferrin, collagenase, complement activator, complement receptor,N-formylmethionyl-leucyl-phenylalanine (FMLP) receptor, lamininreceptor, cytochrome b₅₅₈, monocyte-chemotactic factor, histaminase,vitamin B12 binding protein, gelatinase, plasminogen activator,beta-D-glucuronidase, and a combination thereof. Preferably, theseinflammatory mediators are selected from the group consisting ofmyeloperoxidase (MPO), eosinophil peroxidase (EPO), major basic protein(MBP), lysozyme, granzyme and a combination thereof.

The present invention contacts an effective amount of the peptide withan inflammatory cell, wherein the effective amount is defined as adegranulation-inhibiting amount of MANS peptide or an active fragmentthereof that reduces the amount of an inflammatory mediator releasedfrom at least one inflammatory cell from about 1% to about 99% ascompared to the amount released from at least one inflammatory cell inthe absence of MANS peptide or an active fragment thereof. This amountis also known as an effective inflammatory mediator release-reducingamount. More preferably, this effective amount of the contacted peptidecomprises a degranulation-inhibiting amount of MANS peptide or an activefragment thereof that reduces the amount of an inflammatory mediatorreleased from at least one inflammatory cell from between about 5-50% toabout 99% as compared to the amount released from at least oneinflammatory cell in the absence of MANS peptide or an active fragmentthereof.

The present invention in one embodiment is directed to theadministration of at least one peptide comprising a MANS peptide and anactive fragment thereof in a therapeutically effective amount intotissue or fluid of a subject where the subject is afflicted by arespiratory disease, which is preferably asthma, chronic bronchitis orCOPD. In a further embodiment, the subject may be afflicted by a boweldisease, a skin disease, an autoimmune disease, a pain syndrome, andcombinations thereof. The bowel disease may be ulcerative colitis,Crohn's disease or irritable bowel syndrome. The subject may beafflicted with a skin disease, such as rosacea, eczema, psoriasis orsevere acne. The subject may also be afflicted with arthritis, such asrheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus.Subjects afflicted by cystic fibrosis may also be treated by the presentmethod and peptides. The present method is preferably useful for thetreatment of subjects, such as mammals, and preferably humans, canines,equines and felines.

The present method of treatment of subjects is by the administration ofone or more peptides including the MANS peptide or an active fragmentdescribed herein to include topical administration, parenteraladministration, rectal administration, pulmonary administration, nasaladministration, or oral administration. More specifically, pulmonaryadministration is selected from the group of aerosol, dry powderinhaler, metered dose inhaler, and nebulizer. Additionally, thedisclosed method may further comprise the administration to the subjectof a second molecule selected from the group consisting of anantibiotic, an antiviral compound, an antiparasitic compound, ananti-inflammatory compound, and an immunomodulator.

In one aspect, the invention relates to a method of administering apharmaceutical composition. The pharmaceutical composition comprises atherapeutically effective amount of a known compound and apharmaceutically acceptable carrier. A “therapeutically effective”amount as used herein is an amount of a compound that is sufficient toameliorate symptoms exhibited by a subject. The therapeuticallyeffective amount will vary with the age and physical condition of thepatient, the severity of the condition of the patient being treated, theduration of the treatment, the nature of any concurrent treatment, thepharmaceutically acceptable carrier used and like factors within theknowledge and expertise of those skilled in the art. Pharmaceuticallyacceptable carriers are preferably solid dosage forms such as tablets orcapsules. Liquid preparations for oral administration also may be usedand may be prepared in the form of syrups or suspensions, e.g.,solutions containing an active ingredient, sugar, and a mixture ofethanol, water, glycerol, and propylene glycol. If desired, such liquidpreparations may include one or more of following: coloring agents,flavoring agents, and saccharin. Additionally, thickening agents such ascarboxymethylcellulose also may be used as well as other acceptablecarriers, the selection of which are known in the art.

As stated above, the present invention relates to methods for regulatingcellular secretory processes, especially those releasing inflammatorymediators from inflammatory cells. As used herein, the term “regulating”means blocking, inhibiting, decreasing, reducing, increasing, enhancingor stimulating. A number of cellular secretory processes involve therelease of contents from membrane-bound vesicles or granules withincells A membrane-bound vesicle or granule is defined as an intracellularparticle, which is primarily vesicular (or a vesicle inside a cell) andwhich contains stored material that can be secreted. Some of thecontents of these vesicles, such as those contained in inflammatorycells, have been found to be responsible for a variety of pathologies innumerous mammalian tissues. Some of the effects of these secretionsappear to include damage of previously healthy tissue duringinflammation. This invention provides a means of blocking secretion fromany membrane-bound vesicle, including those found in inflammatory cells,by targeting a specific molecule important in the intracellularsecretory pathway with a synthetic peptide. This approach may be oftherapeutic importance for the treatment of a wide variety ofhypersecretory and inflammatory conditions in humans and animals.

More specifically, the present invention targets inflammatory cells thatcontain the inflammatory mediators in one or more granules or vesicleswithin the cells' cytoplasm. The cells are contacted with one or morepeptides that are selected from the MANS peptide or an active fragmentthereof, all of which are described in detail herein. Preferably thecontact of the peptide with the inflammatory cell is via administrationto a subject afflicted by or suffering from a disease in which theseinflammatory cells are present in specific tissue or fluid within thetissue. Upon administration or contact of the peptide with the cell, thepeptide competitively competes for and competitively inhibits thebinding of the native MARCKS protein to the membrane of theintracellular granules or vesicles which contain the inflammatorymediators. As a result of blocking the binding of the MARCKS protein tothe vesicles in the inflammatory cells, these vesicles in these cells donot move to the plasma membrane of the cells as they would normally dowhen stimulated to exocytotically release their contents of inflammatorymediators out of the cells. Thus, the method of the present inventioninhibits the movement of the vesicles to the cells' plasma membrane,which in turn, reduces the release of the inflammatory mediators fromthe inflammatory cells. The amount of inflammatory mediators releasedfrom the cells over time is reduced because both the rate of release andthe amount of release of the mediators from the inflammatory cells isdependent upon the concentration of the peptide administered andcontacted with the inflammatory cells.

One benefit of the present invention is that it may combine a therapythat includes the direct blocking of mucus secretion with a uniqueanti-inflammatory therapy. A benefit of the present invention overcurrent anti-inflammation therapies that affect a general suppression ofthe immune system is that the peptide is thought to block secretion ofonly intracellular components secreted from inflammatory cells. Thus,many aspects of the immune system should still function even with theinhibition of the inflammatory mediators.

The compounds of the invention may regulate, i.e. block, inflammatorymediator release from cells. This inhibition of release of inflammatorymediators is an attractive means for preventing and treating a varietyof disorders, e.g., diseases and pathological conditions involvinginflammation. Thus, the compounds of the invention may be useful for thetreatment of such conditions. These encompass airway diseases andchronic inflammatory diseases including, but not limited to,osteoarthritis, multiple sclerosis, Guillain-Barre syndrome, Crohn'sdisease, ulcerative colitis, psoriasis, graft versus host disease andsystemic lupus erythematosus. The compounds of the invention can also beused to treat other disorders associated with the activity of elevatedlevels of proinflammatory mediators and enzymes such as responses tovarious infectious agents and a number of diseases of autoimmunity suchas rheumatoid arthritis, toxic shock syndrome, diabetes and inflammatorybowel diseases.

Uses of the peptide and methods of the invention include therapies tocombat inflammation along with therapies that will combine theanti-inflammatory activity of the peptide with its ability to blockmucus secretion. Diseases that may be treated by the peptide's abilityto block both inflammation and mucus secretion include but are notlimited to inflammatory bowel diseases, digestive disorders (i.e.,inflamed gall bladder, Menetier's disease) and inflammatory airwaydiseases.

Other proinflammatory mediators have been correlated with a variety ofdisease states that correlate with influx of neutrophils into sites ofinflammation or injury. Blocking antibodies have been demonstrated asuseful therapies against the neutrophil-associated tissue injury inacute inflammation (Harada et al., 1996, Molecular Medicine Today 2,482). Cells other than neutrophils that may release inflammatorymediators include other leukocytes, such as basophils, eosinophils,monocytes and lymphocytes, and therapies may be directed againstsecretion from these cells. Neutrophils, eosinophils, and basophils areeach a type of granulocyte, i.e., a leukocyte that has granules in itscytoplasm. Leukocytes synthesize a number of inflammatory mediators thatare packaged and stored in cytoplasmic granules. Among these mediatorsare, for example, myeloperoxidase [MPO] in neutrophils (Borregaard N,Cowland J B. Granules of the human neutrophilic polymorphonuclearleukocyte. Blood 1997; 89:3503-3521), eosinophil peroxidase [EPO] andmajor basic protein [MBP] in eosinophils (Gleich G J. Mechanisms ofeosinophil-associated inflammation. J Allergy Clin Immunol 2000;105:651-663), lysozyme in monocytes/macrophages (Hoff T, Spencker T,Emmendoerffer A., Goppelt-Struebe M. Effects of glucocorticoids on theTPA-induced monocytic differentiation. J Leukoc Biol 1992; 52:173-182;Balboa M A, Saez Y, Balsinde J. Calcium-independent phospholipase A2 isrequired for lysozyme secretion in U937 promonocytes. J Immunol 2003;170:5276-5280), and granzyme in natural killer (NK) cells and cytotoxiclymphocytes (Bochan M R, Goebel W S, Brahmi Z. Stably transfectedantisense granzyme B and perforin constructs inhibit humangranule-mediated lytic ability. Cell Immunol 1995; 164:234-239; Gong JH., Maki G, Klingemann H G. Characterization of a human cell line(NK-92) with phenotypical and functional characteristics of activatednatural killer cells. Leukemia 1994; 8:652-658; Maki G, Klingemann H G,Martinson J A, Tam Y K. Factors regulating the cytotoxic activity of thehuman natural killer cell line, NK-92. J Hematother Stem Cell Res 2001;10:369-383; and Takayama H, Trenn G, Sitkovsky M V. A novel cytotoxic Tlymphocyte activation assay. J Immunol Methods 1987; 104:183-1907-10).These mediators can be released at sites of injury and can contribute toinflammation and repair, such as in the lung and elsewhere, as a resultof the infiltration of these cells to the tissue site of injury ordisease. Leukocytes release these granules via an exocytotic mechanism(Burgoyne R D, Morgan A. Secretory granule exocytosis. Physiol Rev 2003;83:581-632; Logan M R, Odemuyiwa S O, Moqbel R. Understanding exocytosisin immune and inflammatory cells: the molecular basis of mediatorsecretion. J Allergy Clin Immunol 2003; 111: 923-932),

Mast cells, which usually do not circulate in the blood stream, andbasophils contain secretory cytoplasmic granules which store and canrelease, upon cell activation, preformed inflammatory (anaphylactic)mediators, such as histamine; proteoglycans, such as heparin andchondroitin sulphate; proteases such as tryptase, chymase,carboxypeptidase, and cathepsin G-like protease; chemotactic factors,cytokines and metabolites of arachidonic acid that act on thevasculature, smooth muscle, connective tissue, mucous glands andinflammatory cells.

Neutrophils, also known as polymorphonuclear leukocytes (PMN), comprise50 to 60% of the total circulating leukocytes. Neutrophils act againstinfectious agents, such as bacteria, fungi, protozoa, viruses, virallyinfected cells, as well as tumor cells, that penetrate the body'sphysical barriers at sites of infection or injury. Neutrophils maturethrough six morphological stages: myeloblast, promyeloblast, myelocyte,metamyelocyte, non-segmented (band) neutrophil, and segmented(functionally active) neutrophil.

In neutrophils, inflammatory mediators are stored in primary(azurophil), secondary (specific), and tertiary (gelatinase) granules,as well as in secretory vesicles. Among numerous mediators ofinflammation, primary (azurophil) granules contain myeloperoxidase(MPO), lysozyme, defensins, bactericidal permeability-increasing protein(BPI), elastase, cathepsin G, cathepsin B, cathepsin D,beta-D-glucuronidase, alpha-mannosidase, phospholipase A₂,chondroitin-4-sulphate, and proteinase 3 (see, for example, Hartwig J H,Thelen M, Rosen A, Janmey P A, Nairn A C, Aderem A. MARCKS is an actinfilament crosslinking protein regulated by protein kinase C andcalcium-calmodulin. Nature 1992; 356:618-622); secondary (specific)granules contain lysozyme, lactoferrin, collagenase, complementactivator, phospholipase A₂, complement receptors, e.g., CR3, CR4,N-formylmethionyl-leucyl-phenylalanine (FMLP) receptors, lamininreceptors, cytochrome b₅₅₈, monocyte-chemotactic factor, histaminase,and vitamin B12 binding protein; and small storage granules containgelatinase, plasminogen activator, cathepsin B, cathepsin D,beta-D-glucuronidase, alpha-mannosidase, and cytochrome b₅₅₈.

Neutrophil granules contain antimicrobial or cytotoxic substances,neutral proteinases, acid hydrolases and a pool of cytoplasmic membranereceptors. Among azurophil granule constituents myeloperoxidase (MPO) isa critical enzyme in the conversion of hydrogen peroxide to hypochlorousacid. Together with hydrogen peroxide and a halide cofactor it forms aneffective microbicidal and cytotoxic mechanism of leukocytes—themyeloperoxidase system.

Defensins, which constitute 30 to 50% of azurophilic granule protein,are small (molecule weight<4000) potent antimicrobial peptides that arecytotoxic to a broad range of bacteria, fungi and some viruses. Theirtoxicity may be due to membrane permeabilization of the target cellwhich is similar to other channel-forming proteins (perforins).

Bacterial permeability-increasing (BPI) protein is a member ofperforins. It is highly toxic to gram-negative bacteria but not togram-positive bacteria or fungi and can also neutralize endotoxin, thetoxic lipopolysaccharide component of gram-negative bacterial cellenvelope.

Lactoferrin sequesters free iron, thereby preventing the growth ofingested microorganisms that survive the killing process and increasesbacterial permeability to lysozyme.

Serine proteases such as elastase and cathepsin G hydrolyze proteins inbacterial cell envelopes. Substrates of granulocyte elastase includecollagen cross-linkages and proteoglycans, as well as elastin componentsof blood vessels, ligaments, and cartilage. Cathepsin D cleavescartilage proteoglycans, whereas granulocyte collagenases are active incleaving type I and, to a lesser degree, type III collagen from bone,cartilage, and tendon. Collagen breakdown products have chemotacticactivity for neutrophils, monocytes, and fibroblasts.

Regulation of tissue destructive potential of lysosomal proteases ismediated by protease inhibitors such as alpha2-macroglobulin andalpha1-antiprotease. These antiproteases are present in serum andsynovial fluids. They may function by binding to and covering the activesites of proteases. Protease-antiprotease imbalance can be important inthe pathogenesis of emphysema.

Azurophil granules function predominantly in the intracellular milieu(in the phagolysosomal vacuole), where they are involved in the killingand degradation of microorganisms. Neutrophil specific granules aresusceptible to release their contents extracellularly and have animportant role in initiating inflammation. Specific granules representan intracellular reservoir of various plasma membrane componentsincluding cytochrome b (component of NADPH oxidase, an enzymeresponsible for the production of superoxide), receptors for complementfragment iC3b (CR3, CR4), for laminin, and formylmethionyl-peptidechemoattractants. In addition to others, there is histaminase which isrelevant for the degradation of histamine, vitamin binding protein, andplasminogen activator which is responsible for plasmin formation andcleavage of C5a from C5.

The importance of neutrophil granules in inflammation is apparent fromstudies of several patients with congenital abnormalities of thegranules. Patients with Chédiak-Higashi syndrome have a profoundabnormality in the rate of establishment of an inflammatory response andhave abnormally large lysosomal granules. The congenital syndrome ofspecific granule deficiency is an exceedingly rare disordercharacterized by diminished inflammatory responses and severe bacterialinfections of skin and deep tissues.

Although mechanisms regulating exocytotic secretion of these granulesare only partially understood, several key molecules in the process havebeen identified, including intracellular Ca2+ transients (Richter et al.Proc Natl Acad Sci USA 1990; 87:9472-9476; Blackwood et al., Biochem J1990; 266:195-200), G proteins, tyrosine and protein kinases (PK,especially PKC) (Smolen et al., Biochim Biophys Acta 1990; 1052:133-142;Niessen et al., Biochim. Biophys. Acta 1994; 1223:267-273; Naucler etal., Pettersen et al., Chest 2002; 121; 142-150), Rac2 (Abdel-Latif etal., Blood 2004; 104:832-839; Lacy et al., J Immunol 2003;170:2670-2679) and various SNARE's, SNAP's and VAMP's (Sollner et al.,Nature 1993; 362: 318-324; Lacy, Pharmacol Ther 2005; 107:358-376).

SNARE (Soluble N-ethylmaleimide attachment protein receptor) proteinsare a family of membrane-associated proteins characterized by analpha-helical coiled-coil domain called the SNARE motif (Li et al.,Cell. Mol. Life Sci. 60: 942-960 (2003)). These proteins are classifiedas v-SNAREs and t-SNAREs based on their localization on vesicle ortarget membrane; another classification scheme defines R-SNAREs andQ-SNAREs, as based on the conserved arginine or glutamine residue in thecentre of the SNARE motif. SNAREs are localized to distinct membranecompartments of the secretory and endocytic trafficking pathways, andcontribute to the specificity of intracellular membrane fusionprocesses. The t-SNARE domain consists of a 4-helical bundle with acoiled-coil twist. The SNARE motif contributes to the fusion of twomembranes. SNARE motifs fall into four classes: homologues of syntaxin1a (t-SNARE), VAMP-2 (v-SNARE), and the N- and C-terminal SNARE motifsof SNAP-25. One member from each class may interact to form a SNAREcomplex. The SNARE motif is found in the N-terminal domains of certainsyntaxin family members such as syntaxin 1a, which is required forneurotransmitter release (Lerman et al., Biochemistry 39: 8470-8479(2000)), and syntaxin 6, which is found in endosomal transport vesicles(Misura et al., Proc. Natl. Acad. Sci. U.S.A. 99: 9184-9189 (2002)).

SNAP-25 (synaptosome-associated protein 25 kDa) proteins are componentsof SNARE complexes, which may account for the specificity of membranefusion and to directly execute fusion by forming a tight complex (theSNARE or core complex) that brings the synaptic vesicle and plasmamembranes together. The SNAREs constitute a large family of proteinsthat are characterized by 60-residue sequences known as SNARE motifs,which have a high propensity to form coiled coils and often precedecarboxy-terminal transmembrane regions. The synaptic core complex isformed by four SNARE motifs (two from SNAP-25 and one each fromsynaptobrevin and syntaxin 1) that are unstructured in isolation butform a parallel four-helix bundle on assembly. The crystal structure ofthe core complex has revealed that the helix bundle is highly twistedand contains several salt bridges on the surface, as well as layers ofinterior hydrophobic residues. A polar layer in the centre of thecomplex is formed by three glutamines (two from SNAP-25 and one fromsyntaxin 1) and one arginine (from synaptobrevin) (Rizo et al., Nat RevNeurosci 3: 641-653 (2002)). Members of the SNAP-25 family contain acluster of cysteine residues that can be palmitoylated for membraneattachment (Risinger et al., J. Biol. Chem. 268: 24408-24414 (1993)).

The major role of neutrophils is to phagocytose and destroy infectiousagents. They also limit the growth of some microbes, prior to onset ofadaptive (specific) immunological responses. Although neutrophils areessential to host defense, they have also been implicated in thepathology of many chronic inflammatory conditions and inischemia-reperfusion injury. Hydrolytic enzymes of neutrophil origin andoxidatively inactivated protease inhibitors can be detected in fluidisolated from inflammatory sites. Under normal conditions, neutrophilscan migrate to sites of infection without damage to host tissues.However, undesirable damage to a host tissue can sometimes occur. Thisdamage may occur through several independent mechanisms. These includepremature activation during migration, extracellular release of toxicproducts during the killing of some microbes, removal of infected ordamage host cells and debris as a first step in tissue remodeling, orfailure to terminate acute inflammatory responses. Ischemia-reperfusioninjury is associated with an influx of neutrophils into the affectedtissue and subsequent activation. This may be triggered by substancesreleased from damaged host cells or as a consequence of superoxidegeneration through xantine oxidase.

Under normal conditions, blood may contain a mixture of normal, primed,activated and spent neutrophils. In an inflammatory site, mainlyactivated and spent neutrophils are present. Activated neutrophils haveenhanced production of reactive oxygen intermediates (ROI). Asubpopulation of neutrophils with the enhanced respiratory burst hasbeen detected in the blood of people with an acute bacterial infectionand patients with the adult respiratory distress syndrome (ARDS). Thisis an example of a neutrophil paradox. Neutrophils have been implicatedin the pathology of this condition because of the large influx of thesecells into the lung and the associated tissue damage caused by oxidantsand hydrolytic enzymes released from activated neutrophils. Theimpairment of neutrophil microbicidal activity that occurs as the ARDSworsens may be a protective response on the part of the host, which isinduced locally by inflammatory products.

The acute phase of thermal injury is also associated with neutrophilactivation, and this is followed by a general impairment in variousneutrophil functions. Activation of neutrophils by immune complexes insynovial fluid contributes to the pathology of rheumatoid arthritis.Chronic activation of neutrophils may also initiate tumor developmentbecause some ROI generated by neutrophils damage DNA and proteasespromote tumor cell migration. In patients suffering from severe burns, acorrelation has been established between the onset of bacterialinfection and reduction in the proportion and absolute numbers ofneutrophils positive for antibody and complement receptors. Oxidants ofneutrophil origin have also been shown to oxidize low-densitylipoproteins (LDL), which are then more effectively bound to the plasmamembrane of macrophages through specific scavenger receptors. Uptake ofthese oxidized LDL by macrophages may initiate atherosclerosis. Inaddition, primed neutrophils have been found in people with essentialhypertension, Hodgkin's disease, inflammatory bowel disease, psoriasis,sarcoidosis, and septicemia, where priming correlates with highconcentrations of circulating TNF-alpha (cachectin).

Hydrolytic damage to host tissue and therefore chronic inflammatoryconditions may occur when antioxidant and antiprotease screens areoverwhelmed. Antiprotease deficiency is thought to be responsible forthe pathology of emphysema. Many antiproteases are members of the serineprotease inhibitor (SERPIN) family. Although the circulation is rich inantiproteases, these large proteins may be selectively excluded at sitesof inflammation because neutrophils adhere to their targets. Oxidativestress may initiate tissue damage by reducing the concentration ofextracellular antiproteases to below the level required to inhibitreleased proteases. Chlorinated oxidants and hydrogen peroxide caninactivate antiproteases such as alpha1-protease inhibitor andalpha2-macroglobulin, which are endogenous inhibitors of elastase, butsimultaneously activate latent metalloproteases such as collagenases andgelatinase, which contribute to the further inactivation ofantiproteases.

Cytoplasmic constituents of neutrophils may also be a cause of formationof specific anti-neutrophil cytoplasmic antibodies (ANCA), which areclosely related to the development of systemic vasculitis andglomerulonephritis. ANCA are antibodies directed against enzymes thatare found mainly within the azurophil or primary granules ofneutrophils. There are three types of ANCA that can be distinguished bythe patterns they produce by indirect immunofluorescence on normalethanol-fixed neutrophils. Diffuse fine granular cytoplasmicfluorescence (cANCA) is typically found in Wegener's granulomatosis, insome cases of microscopic polyarteritis and Churg Strauss syndrome, andin some cases of crescentic and segmental necrotizingglomerulonephritis. The target antigen is usually proteinase 3.Perinuclear fluorescence (pANCA) is found in many cases of microscopicpolyarteritis and glomerulonephritis. These antibodies are oftendirected against myeloperoxidase but other targets include elastase,cathepsin G, lactoferrin, lysozyme and beta-D-glucuronidase. The thirdgroup designated “atypical” ANCA includes neutrophil nuclearfluorescence and some unusual cytoplasmic patterns and while a few ofthe target antigens are shared with pANCA, the others have not beenidentified yet. pANCA are also found in a third of patients with Crohn'sdisease. The reported incidence of ANCA in rheumatoid arthritis and SLEvaries considerably but the patterns are predominantly pANCA andatypical ANCA.

The eosinophil is a terminally differentiated, end-stage leukocyte thatresides predominantly in submucosal tissue and is recruited to sites ofspecific immune reactions, including allergic diseases. The eosinophilcytoplasm contains large ellipsoid granules with an electron-densecrystalline nucleus and partially permeable matrix. In addition to theselarge primary crystalloid granules, there is another granule type thatis smaller (small granule) and lacks the crystalline nucleus. The largespecific granules of eosinophils contain at least four distinct cationicproteins, which exert a range of biological effects on host cells andmicrobial targets: major basic protein (MBP), eosinophil cationicprotein (ECP), eosinophil derived neurotoxin (EDN), and eosinophilperoxidase (EPO). Basophils contain about one fourth as much major basicprotein as eosinophils together with detectable amounts of EDN, ECP andEPO. Small amounts of EDN and ECP are also found in neutrophils (GleichG J. Mechanisms of eosinophil-associated inflammation. J Allergy ClinImmunol 2000; 105:651-663). MBP appears to lack enzymatic activity butis a highly cationic polypeptide which may exert its toxic activities byinteractions with lipid membranes leading to their derangement. Both MBPand EPO can act as selective allosteric inhibitors of agonist binding toM2 muscarinic receptors. These proteins may contribute to M2 receptordysfunction and enhance vagally mediated bronchoconstriction in asthma.EDN can specifically damage the myelin coat of neurons. Histaminase anda variety of hydrolytic lysosomal enzymes are also present in the largespecific granules of eosinophils. Among the enzymes in small granules ofeosinophils are aryl sulphatase, acid phosphatase, and a 92 kDametalloproteinase, a gelatinase. Eosinophils can elaborate cytokineswhich include those with potential autocrine growth-factor activitiesfor eosinophils and those with potential roles in acute and chronicinflammatory responses. Three cytokines have growth-factor activitiesfor eosinophils: granulocyte-macrophage colony-stimulating factor(GM-CSF), IL-3 and IL-5. Other cytokines produced by human eosinophilsthat may have activities in acute and chronic inflammatory responsesinclude IL-1-alpha, IL-6, IL-8, TNF-alpha and both transforming growthfactors, TGF-alpha and TGF-beta.

Eosinophils contain crystalloid granules that contain MBP, eosinophilcationic protein, EPO, and eosinophil-derived neurotoxin (Gleich, JAllergy Clin Immunol 2000; 105:651-663). The human promyelocytic cellline HL-60 clone 15 can be used to examine secretion of EPO. This cellline was established from a clone of HL-60 that had been grown at anelevated pH for two months (Fischkoff, Leuk Res 1988; 12:679-686) andthen treated with butyric acid to allow the cells to differentiate so asto exhibit many of the characteristics of peripheral blood eosinophils,including expression of eosinophil-specific granule proteins (Rosenberget al., J Exp Med 1989; 170:163-176; Tiffany et al., J Leukoc Biol 1995;58:49-54; Badewa et al., Exp Biol Med 2002; 227:645-651).

Eosinophils can participate in hypersensitivity reactions, especiallythrough two lipid inflammatory mediators, leukotriene C⁴ (LTC⁴) andplatelet activating factor (PAF). Both mediators contract airway smoothmuscle, promote the secretion of mucus, alter vascular permeability andelicit eosinophil and neutrophil infiltration. In addition to the directactivities of these eosinophil-derived mediators, MBP can stimulate therelease of histamine from basophils and mast cells, and MBP canstimulate the release of EPO from mast cells. Eosinophils can serve as alocal source of specific lipid mediators as well as induce the releaseof mediators from mast cells and basophils. Eosinophil granule contentis released following similar stimuli to neutrophil granules, e.g.during phagocytosis of opsonized particles and by chemotactic factors.Neutrophil lysosomal enzymes act primarily on material engulfed inphagolysosomes, while the eosinophil granule contents act mainly onextracellular target structure such as parasites and inflammatorymediators.

Monocyte and macrophage development takes place in the bone marrow andpasses through the following steps: stem cell; committed stem cell;monoblast; promonocyte; monocyte in bone marrow; monocyte in peripheralblood; and macrophage in tissues. Monocyte differentiation in the bonemarrow proceeds rapidly (1.5 to 3 days). During differentiation,granules are formed in monocyte cytoplasm and these can be divided as inneutrophils into at least two types. However, they are fewer and smallerthan their neutrophil counterparts (azurophil and specific granules).Their enzyme content is similar.

Granule-bound enzymes of monocytes/macrophages include lysozyme, acidphosphatase, and beta-glucuronidase. As a model for in vivo studies,lysozyme secretion from U937 cells was used. This cell line is derivedfrom a human histiocytic lymphoma and has been used as a monocytic cellline that can be activated by a variety of agonists, such as PMA (Hoffet al., J Leukoc Biol 1992; 52:173-182; Balboa et al., J Immunol 2003;170:5276-5280; Sundstrom et al., Int J Cancer 1976; 17:565-577).

Natural killer (NK) cells and cytotoxic lymphocytes contain potentcytotoxic granules including perforin, a pore-forming protein, andgranzymes, lymphocyte-specific serine proteases. For example, the NK-92cell line is an IL-2-dependent human line established from a patientwith rapidly progressive non-Hodgkin's lymphoma (Gong J H., Maki G,Klingemann H G. Characterization of a human cell line (NK-92) withphenotypical and functional characteristics of activated natural killercells. Leukemia 1994; 8:652-658). NK-92 cells express high levels ofmolecules involved in the perforin-granzyme cytolytic pathway thattargets a wide range of malignant cells (Gong et al, vide infra, andMaki G, Klingemann H G, Martinson J A, Tam Y K. Factors regulating thecytotoxic activity of the human natural killer cell line, NK-92. JHematother Stem Cell Res 2001; 10:369-383).

Granzymes are exogenous serine proteases that are released bycytoplasmic granules within cytotoxic T cells and natural killer cells.Granzymes can induce apoptosis within virus-infected cells, thusdestroying them.

Extracellular release of a mediator of inflammation (inflammatorymediator) from a granulocyte (or leukocyte), and extracellular releaseof more than one mediator of inflammation (inflammatory mediator) from agranulocyte (or leukocyte) is sometimes referred to herein asdegranulation. In a preferred embodiment, the release of a mediator ofinflammation comprises release of said mediator from a granule locatedin the interior of a granulocyte or leukocyte. The release ofinflammatory mediator is preferably the release of an inflammatorymediator from these granules.

Neutrophils and macrophages, upon priming by pro-inflammatory agents(inflammatory stimulants) such as TNFα, dramatically increase theirsynthesis of MARCKS protein: as much as 90% of the new protein formed byneutrophils in response to either TNFα or lipopolysaccharide (LPS) isMARCKS (Thelen M, Rosen A, Nairn A C, Aderem A. Tumor necrosis factoralpha modifies agonist-dependent responses in human neutrophils byinducing the synthesis and myristoylation of a specific protein kinase Csubstrate. Proc Natl Acad Sci USA 1990; 87:5603-5607). MARCKS can thushave an important role in subsequent release of inflammatory mediatorswhen granule-containing cells, such as neutrophils and macrophages, arestimulated by agonists, especially those that work by activating PKC(Burgoyne et al., Physiol Rev 2003; 83:581-632; Logan et al. J AllergyClin Immunol 2003; 111: 923-932; Smolen et al., Biochim Biophys Acta1990; 1052:133-142; Niessen et al., Biochim. Biophys. Acta 1994;1223:267-273; Naucler et al., J Leukoc Biol 2002; 71:701-710).

In one aspect of this invention, administration of adegranulation-inhibiting amount of MANS peptide or an active fragmentthereof as described herein to a site of inflammation in a subject,which site of inflammation has resulted from the onset of entry of adisease, a condition, a trauma, a foreign body, or a combination thereofat the site of inflammation in the subject, can reduce the amount of amediator of inflammation released from infiltrating leukocytes at thesite of inflammation, where the leukocytes are preferably granulocytes.The administration of the MANS peptide and/or at least one activefragment thereof can reduce the amount of a mediator of inflammationreleased from leukocytes such as granulocytes infiltrating into the siteof inflammation. The degranulation-inhibiting amount of MANS peptide, orthe degranulation-inhibiting amount of an active fragment thereof, issufficient to reduce or inhibit the exocytotic release of inflammatorymediators from granules contained within the inflammatory cellsinfiltrating into the site. Degranulation-inhibiting efficacy ismeasured at a time after administration of the MANS peptide or theactive fragment thereof by comparison of the percent of inhibition(i.e., percent of reduction) of the release of mediators of inflammationfrom said cells (leukocytes or granulocytes or other inflammatory cells)relative to the level or amount or concentration of said mediators ofinflammation released or produced at approximately the same time in theabsence of MANS peptide and/or in the absence of the active fragmentthereof. Additionally, a skilled clinician can determine whetherinflammation at the tissue site has been reduced by measuring symptomsand parameters of inflammation known as indicators of the disease todetermine whether a sufficient or therapeutically effective amount MANSpeptide and/or an active fragment thereof has been administered. Asufficient degranulation-inhibiting amount is the amount which producesa percentage of reduction of a mediator of inflammation released from agranulocyte, at the site of inflammation, which percentage is from about1% to about 99%, preferably from 5% to about 99%, more preferably fromabout 10% to about 99%, even more preferably from about 25% to 99%, andeven more preferably from about 50% to about 99% of the amount of saidmediator of inflammation released from said granulocyte in the absenceof MANS peptide or an active fragment thereof tested under the sameconditions.

In one aspect of this invention, administration of adegranulation-inhibiting amount of MANS peptide to a site ofinflammatory stimulation in an animal, which site of inflammatorystimulation has been created by administration of aninflammation-stimulating amount of an inflammatory stimulant to saidsite, can reduce the amount of a mediator of inflammation released froma granulocyte, which granulocyte is stimulated by said inflammatorystimulant at said site of inflammatory stimulation, from about 1% toabout 99%, preferably from 5% to about 99%, more preferably from about10% to about 99%, even more preferably from about 25% to 99%, and evenmore preferably from about 50% to about 99% of the amount of saidmediator of inflammation released from said granulocyte in the absenceof MANS peptide in the presence of the identicalinflammation-stimulating amount of said inflammatory stimulant.

In another aspect of this invention, administration of adegranulation-inhibiting amount of MANS peptide to a site ofinflammatory stimulation in an animal, which site of inflammatorystimulation has been created by administration of aninflammation-stimulating amount of an inflammatory stimulant to saidsite, can reduce the amount of a mediator of inflammation released froma granulocyte, which granulocyte is stimulated by said inflammatorystimulant at said site of inflammatory stimulation, by 100% of theamount of said mediator of inflammation released from said granulocytein the absence of MANS peptide in the presence of the identicalinflammation-stimulating amount of said inflammatory stimulant.

An example of an inflammatory stimulant used in in vitro examples hereinis phorbol 12-myristate 13-acetate (PMA). Monocyte chemoattractantprotein (MCP-1) is nearly as effective as C5a, and much more potent thanIL-8, in the degranulation of basophils, resulting in histamine release.Histamine release can occur after stimulation with chemokines (i.e.,chemoattractant cytokines), RANTES and MIP-1.

In a preferred embodiment, relative to the basal concentration of MARCKSpeptide present at the site of inflammatory stimulation, thedegranulation-inhibiting amount of MANS peptide administered to a siteof inflammatory stimulation in an animal comprises from about 1 time toabout 1,000,000 times the concentration of the MARCKS peptide at saidsite of inflammatory stimulation, preferably from about 1 time to about100,000 times the concentration of the MARCKS peptide at said site ofinflammatory stimulation, more preferably from about 1 time to about10,000 times the concentration of the MARCKS peptide at said site ofinflammatory stimulation, even more preferably from about 1 time toabout 1,000 times the concentration of the MARCKS peptide at said siteof inflammatory stimulation, even more preferably from about 1 time toabout 100 times the concentration of the MARCKS peptide at said site ofinflammatory stimulation, and even more preferably from about 1 time toabout 10 times the concentration of the MARCKS peptide at said site ofinflammatory stimulation.

In a preferred embodiment, the granulocyte resides on or in the airwayof an animal, preferably a human, and the MANS peptide is administeredby inhalation, such as by inhalation of a pharmaceutical compositioncomprising the MANS peptide, for example a pharmaceutical compositioncomprising the MANS peptide and an aqueous solution, which compositionis administered in the form of an aerosol, or a pharmaceuticalcomposition comprising the MANS peptide in the form of a dry powder,which composition is administered using a dry powder inhaler. Othermethods and devices known in the art for administration of a solution orpowder by inhalation such as, for example, droplets, sprays, andnebulizers, can be useful.

In some embodiments, it is possible that the peptide of the presentinvention may block secretory processes that are physiologicallyimportant, including basal secretory functions. Although inventors donot wish to be bound to any particular theory of the invention, it isthought that the mechanisms regulating such basal secretion aredifferent than those regulating stimulated secretion. Alternatively,basal secretory mechanisms may require less MARCKS protein thanstimulated secretion. Basal secretion may be preserved since alltherapies to block MARCKS-mediated secretion may not eliminate allMARCKS function.

As used herein, the term “MARCKS nucleotide sequence” refers to anynucleotide sequence derived from a gene encoding a MARCKS protein,including, for example, DNA or RNA sequence, DNA sequence of the gene,any transcribed RNA sequence, RNA sequence of the pre-mRNA or mRNAtranscript, and DNA or RNA bound to protein.

Precise delivery of the MARCKS-blocking peptide may also overcome anypotential limitations of blocking important secretory processes.Delivering such agents to the respiratory tract should be readilyaccomplished with inhaled formulations. Since these agents may be usefulin treating inflammatory bowel disease, one can envision delivery of theblocking agents into the rectum/colon/intestinal tract via enema orsuppositories. Intraarticular injections or transdermal delivery intoinflamed joints may yield relief to patients with arthritic orautoimmune diseases by limiting the secretion from localizedinflammatory cells. Injection into areas surrounding nerve endings mayinhibit secretion of some types of neurotransmitters, blockingtransmission of severe pain or uncontrolled muscle spasms. Delivery ofthe peptide for the treatment of inflammatory skin diseases should bereadily accomplished using various topical formulations known in theart.

It is believed that MARCKS interacts with actin and myosin in thecytoplasm and thus may be able to tether the granules to the cellularcontractile apparatus, thus, mediating subsequent granule movement andexocytosis. Secretion of the inflammatory mediatory MPO from neutrophilsmay also be maximized by activation of both PKC and PKG. It is possiblethat MARCKS serves as the point of convergence for coordinating actionsof these two protein kinases that control secretion from membrane-boundcompartments in inflammatory cells (i.e. secretion of MPO fromneutrophils).

The present invention demonstrates secretion of the inflammatorymediator MPO from canine or human neutrophils was enhanced by concurrentactivation of both PKC and PKG, while activation of either kinase alonewas insufficient to induce a maximal secretory response. An enhancedsecretory response to PMA alone has been documented in NHBE cells and inneutrophils as demonstrated herein, although the magnitude of theresponse was much less than that observed by others in a rat goblet-likecell line. See, Abdullah et al, supra. In addition, although it wasreported previously that a cGMP analogue could induce significant mucinsecretion from cultured guinea pig tracheal epithelial cells (Fischer etal., supra), it should be noted that this response did not reachsignificant levels until 8 h of exposure. A secretory response with sucha long lag period is unlikely to be a direct effect and probablyinvolves de novo protein synthesis as opposed to release of preformedand stored cytoplasmic granules.

As stated above, the present invention may be used in a pharmaceuticalformulation. In certain embodiments, the drug product is present in asolid pharmaceutical composition that may be suitable for oraladministration. A solid composition of matter according to the presentinvention may be formed and may be mixed with and/or diluted by anexcipient. The solid composition of matter also may be enclosed within acarrier, which may be, for example, in the form of a capsule, sachet,tablet, paper, or other container. When the excipient serves as adiluent, it may be a solid, semi-solid, or liquid material that acts asa vehicle, carrier, or medium for the composition of matter.

Various suitable excipients will be understood by those skilled in theart and may be found in the National Formulary, 19: 2404-2406 (2000),the disclosure of pages 2404 to 2406 being incorporated herein in theirentirety. Examples of suitable excipients include, but are not limitedto, starches, gum arabic, calcium silicate, microcrystalline cellulose,methacrylates, shellac, polyvinylpyrrolidone, cellulose, water, syrup,and methylcellulose. The drug product formulations additionally caninclude lubricating agents such as, for example, talc, magnesiumstearate and mineral oil; wetting agents; emulsifying and suspendingagents; preserving agents such as methyl- and propyl hydroxybenzoates;sweetening agents; or flavoring agents. Polyols, buffers, and inertfillers also may be used. Examples of polyols include, but are notlimited to, mannitol, sorbitol, xylitol, sucrose, maltose, glucose,lactose, dextrose, and the like. Suitable buffers include, but are notlimited to, phosphate, citrate, tartrate, succinate, and the like. Otherinert fillers that may be used include those that are known in the artand are useful in the manufacture of various dosage forms. If desired,the solid formulations may include other components such as bulkingagents and/or granulating agents, and the like. The drug products of theinvention may be formulated so as to provide quick, sustained, ordelayed release of the active ingredient after administration to thepatient by employing procedures well known in the art.

To form tablets for oral administration, the composition of matter ofthe present invention may be made by a direct compression process. Inthis process, the active drug ingredients may be mixed with a solid,pulverant carrier such as, for example, lactose, saccharose, sorbitol,mannitol, starch, amylopectin, cellulose derivatives or gelatin, andmixtures thereof, as well as with an antifriction agent such as, forexample, magnesium stearate, calcium stearate, and polyethylene glycolwaxes. The mixture may then be pressed into tablets using a machine withthe appropriate punches and dies to obtain the desired tablet size. Theoperating parameters of the machine may be selected by the skilledartisan. Alternatively, tablets for oral administration may be formed bya wet granulation process. Active drug ingredients may be mixed withexcipients and/or diluents. The solid substances may be ground or sievedto a desired particle size. A binding agent may be added to the drug.The binding agent may be suspended and homogenized in a suitablesolvent. The active ingredient and auxiliary agents also may be mixedwith the binding agent solution. The resulting dry mixture is moistenedwith the solution uniformly. The moistening typically causes theparticles to aggregate slightly, and the resulting mass is pressedthrough a stainless steel sieve having a desired size. The mixture isthen dried in controlled drying units for the determined length of timenecessary to achieve a desired particle size and consistency. Thegranules of the dried mixture are sieved to remove any powder. To thismixture, disintegrating, antifriction, and/or anti-adhesive agents maybe added. Finally, the mixture is pressed into tablets using a machinewith the appropriate punches and dies to obtain the desired tablet size.The operating parameters of the machine may be selected by the skilledartisan.

If coated tablets are desired, the above prepared core may be coatedwith a concentrated solution of sugar or cellulosic polymers, which maycontain gum arabic, gelatin, talc, titanium dioxide, or with a lacquerdissolved in a volatile organic solvent or a mixture of solvents. Tothis coating various dyes may be added in order to distinguish amongtablets with different active compounds or with different amounts of theactive compound present. In a particular embodiment, the activeingredient may be present in a core surrounded by one or more layersincluding enteric coating layers.

Soft gelatin capsules may be prepared in which capsules contain amixture of the active ingredient and vegetable oil. Hard gelatincapsules may contain granules of the active ingredient in combinationwith a solid, pulverulent carrier, such as, for example, lactose,saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin,cellulose derivatives, and/or gelatin.

Liquid preparations for oral administration may be prepared in the formof syrups or suspensions, e.g., solutions containing an activeingredient, sugar, and a mixture of ethanol, water, glycerol, andpropylene glycol. If desired, such liquid preparations may comprise oneor more of following: coloring agents, flavoring agents, and saccharin.Thickening agents such as carboxymethylcellulose also may be used.

In the event that the above pharmaceuticals are to be used forparenteral administration, such a formulation may comprise sterileaqueous injection solutions, non-aqueous injection solutions, or both,comprising the composition of matter of the present invention. Whenaqueous injection solutions are prepared, the composition of matter maybe present as a water soluble pharmaceutically acceptable salt.Parenteral preparations may contain anti-oxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the intended recipient. Aqueous and non-aqueous sterilesuspensions may comprise suspending agents and thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample sealed ampules and vials. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

The composition of matter also may be formulated such that it may besuitable for topical administration (e.g., skin cream). Theseformulations may contain various excipients known to those skilled inthe art. Suitable excipients may include, but are not limited to, cetylesters wax, cetyl alcohol, white wax, glyceryl monostearate, propyleneglycol, monostearate, methyl stearate, benzyl alcohol, sodium laurylsulfate, glycerin, mineral oil, water, carbomer, ethyl alcohol, acrylateadhesives, polyisobutylene adhesives, and silicone adhesives.

In a preferred embodiment, peptide fragments are disclosed in Table 2and are of a length of at least 4 to 23 amino acid residues in lengthand having amino acid sequences identical to an amino acid sequence ofthe MANS peptide, wherein the N-terminal amino acid of the peptides areselected from position 2 to 21 of the MANS peptide sequence (SEQ ID NO:1). The more preferred peptide fragment length is from at least 6 aminoacids to 23 amino acids. Preferably these peptides are acylated at thealpha N-terminal amino acid, and more preferably these peptides aremyristoylated at the alpha-N-terminal amino acid position.

TABLE 2 Sequence Peptide No. Sequence ID No. peptide 3AQFSKTAAKGEAAAERPGEAAVA SEQ ID NO. 3 peptide 5 AQFSKTAAKGEAAAERPGEAAVSEQ ID NO. 5 peptide 8 AQFSKTAAKGEAAAERPGEAA SEQ ID NO. 8 peptide 12AQFSKTAAKGEAAAERPGEA SEQ ID NO. 12 peptide 17 AQFSKTAAKGEAAAERPGESEQ ID NO. 17 peptide 23 AQFSKTAAKGEAAAERPG SEQ ID NO. 23 peptide 30AQFSKTAAKGEAAAERP SEQ ID NO. 30 peptide 38 AQFSKTAAKGEAAAERSEQ ID NO. 38 peptide 47 AQFSKTAAKGEAAAE SEQ ID NO. 47 peptide 57AQFSKTAAKGEAAA SEQ ID NO. 57 peptide 68 AQFSKTAAKGEAA SEQ ID NO. 68peptide 80 AQFSKTAAKGEA SEQ ID NO. 80 peptide 93 AQFSKTAAKGESEQ ID NO. 93 peptide 107 AQFSKTAAKG SEQ ID NO. 107 peptide 122AQFSKTAAK SEQ ID NO. 122 peptide 138 AQFSKTAA SEQ ID NO. 138 peptide 155AQFSKTA SEQ ID NO. 155 peptide 173 AQFSKT SEQ ID NO. 173 peptide 192AQFSK SEQ ID NO. 192 peptide 212 AQFS SEQ ID NO. 212 peptide 6QFSKTAAKGEAAAERPGEAAVA SEQ ID NO. 6 peptide 9 QFSKTAAKGEAAAERPGEAAVSEQ ID NO. 9 peptide 13 QFSKTAAKGEAAAERPGEAA SEQ ID NO. 13 peptide 18QFSKTAAKGEAAAERPGEA SEQ ID NO. 18 peptide 24 QFSKTAAKGEAAAERPGESEQ ID NO. 24 peptide 31 QFSKTAAKGEAAAERPG SEQ ID NO. 31 peptide 39QFSKTAAKGEAAAERP SEQ ID NO. 39 peptide 48 QFSKTAAKGEAAAER SEQ ID NO. 48peptide 58 QFSKTAAKGEAAAE SEQ ID NO. 58 peptide 69 QFSKTAAKGEAAASEQ ID NO. 69 peptide 81 QFSKTAAKGEAA SEQ ID NO. 81 peptide 94QFSKTAAKGEA SEQ ID NO. 94 peptide 108 QFSKTAAKGE SEQ ID NO. 108peptide 123 QFSKTAAKG SEQ ID NO. 123 peptide 139 QFSKTAAK SEQ ID NO. 139peptide 156 QFSKTAA SEQ ID NO. 156 peptide 174 QFSKTA SEQ ID NO. 174peptide 193 QFSKT SEQ ID NO. 193 peptide 213 QFSK SEQ ID NO. 213peptide 10 FSKTAAKGEAAAERPGEAAVA SEQ ID NO. 10 peptide 14FSKTAAKGEAAAERPGEAAV SEQ ID NO. 14 peptide 19 FSKTAAKGEAAAERPGEAASEQ ID NO. 19 peptide 25 FSKTAAKGEAAAERPGEA SEQ ID NO. 25 peptide 32FSKTAAKGEAAAERPGE SEQ ID NO. 32 peptide 40 FSKTAAKGEAAAERPGSEQ ID NO. 40 peptide 49 FSKTAAKGEAAAERP SEQ ID NO. 49 peptide 59FSKTAAKGEAAAER SEQ ID NO. 59 peptide 70 FSKTAAKGEAAAE SEQ ID NO. 70peptide 82 FSKTAAKGEAAA SEQ ID NO. 82 peptide 95 FSKTAAKGEAASEQ ID NO. 95 peptide 109 FSKTAAKGEA SEQ ID NO. 109 peptide 124FSKTAAKGE SEQ ID NO. 124 peptide 140 FSKTAAKG SEQ ID NO. 140 peptide 157FSKTAAK SEQ ID NO. 157 peptide 175 FSKTAA SEQ ID NO. 175 peptide 194FSKTA SEQ ID NO. 194 peptide 214 FSKT SEQ ID NO. 214 peptide 15SKTAAKGEAAAERPGEAAVA SEQ ID NO. 15 peptide 20 SKTAAKGEAAAERPGEAAVSEQ ID NO. 20 peptide 26 SKTAAKGEAAAERPGEAA SEQ ID NO. 26 peptide 33SKTAAKGEAAAERPGEA SEQ ID NO. 33 peptide 41 SKTAAKGEAAAERPGESEQ ID NO. 41 peptide 50 SKTAAKGEAAAERPG SEQ ID NO. 50 peptide 60SKTAAKGEAAAERP SEQ ID NO. 60 peptide 71 SKTAAKGEAAAER SEQ ID NO. 71peptide 83 SKTAAKGEAAAE SEQ ID NO. 83 peptide 96 SKTAAKGEAAASEQ ID NO. 96 peptide 110 SKTAAKGEAA SEQ ID NO. 110 peptide 125SKTAAKGEA SEQ ID NO. 125 peptide 141 SKTAAKGE SEQ ID NO. 141 peptide 158SKTAAKG SEQ ID NO. 158 peptide 176 SKTAAK SEQ ID NO. 176 peptide 195SKTAA SEQ ID NO. 195 peptide 215 SKTA SEQ ID NO. 215 peptide 21KTAAKGEAAAERPGEAAVA SEQ ID NO. 21 peptide 27 KTAAKGEAAAERPGEAAVSEQ ID NO. 27 peptide 34 KTAAKGEAAAERPGEAA SEQ ID NO. 34 peptide 42KTAAKGEAAAERPGEA SEQ ID NO. 42 peptide 51 KTAAKGEAAAERPGE SEQ ID NO. 51peptide 61 KTAAKGEAAAERPG SEQ ID NO. 61 peptide 72 KTAAKGEAAAERPSEQ ID NO. 72 peptide 84 KTAAKGEAAAER SEQ ID NO. 84 peptide 97KTAAKGEAAAE SEQ ID NO. 97 peptide 111 KTAAKGEAAA SEQ ID NO. 111peptide 126 KTAAKGEAA SEQ ID NO. 126 peptide 142 KTAAKGEA SEQ ID NO. 142peptide 159 KTAAKGE SEQ ID NO. 159 peptide 177 KTAAKG SEQ ID NO. 177peptide 196 KTAAK SEQ ID NO. 196 peptide 216 KTAA SEQ ID NO. 216peptide 28 TAAKGEAAAERPGEAAVA SEQ ID NO. 28 peptide 35 TAAKGEAAAERPGEAAVSEQ ID NO. 35 peptide 43 TAAKGEAAAERPGEAA SEQ ID NO. 43 peptide 52TAAKGEAAAERPGEA SEQ ID NO. 52 peptide 62 TAAKGEAAAERPGE SEQ ID NO. 62peptide 73 TAAKGEAAAERPG SEQ ID NO. 73 peptide 85 TAAKGEAAAERPSEQ ID NO. 85 peptide 98 TAAKGEAAAER SEQ ID NO. 98 peptide 112TAAKGEAAAE SEQ ID NO. 112 peptide 127 TAAKGEAAA SEQ ID NO. 127peptide 143 TAAKGEAA SEQ ID NO. 143 peptide 160 TAAKGEA SEQ ID NO. 160peptide 178 TAAKGE SEQ ID NO. 178 peptide 197 TAAKG SEQ ID NO. 197peptide 217 TAAK SEQ ID NO. 217 peptide 36 AAKGEAAAERPGEAAVASEQ ID NO. 36 peptide 44 AAKGEAAAERPGEAAV SEQ ID NO. 44 peptide 53AAKGEAAAERPGEAA SEQ ID NO. 53 peptide 63 AAKGEAAAERPGEA SEQ ID NO. 63peptide 74 AAKGEAAAERPGE SEQ ID NO. 74 peptide 86 AAKGEAAAERPGSEQ ID NO. 86 peptide 99 AAKGEAAAERP SEQ ID NO. 99 peptide 113AAKGEAAAER SEQ ID NO. 113 peptide 128 AAKGEAAAE SEQ ID NO. 128peptide 144 AAKGEAAA SEQ ID NO. 144 peptide 161 AAKGEAA SEQ ID NO. 161peptide 179 AAKGEA SEQ ID NO. 179 peptide 198 AAKGE SEQ ID NO. 198peptide 218 AAKG SEQ ID NO. 218 peptide 45 AKGEAAAERPGEAAVASEQ ID NO. 45 peptide 54 AKGEAAAERPGEAAV SEQ ID NO. 54 peptide 64AKGEAAAERPGEAA SEQ ID NO. 64 peptide 75 AKGEAAAERPGEA SEQ ID NO. 75peptide87 AKGEAAAERPGE SEQ ID NO. 87 peptide100 AKGEAAAERPGSEQ ID NO. 100 peptide114 AKGEAAAERP SEQ ID NO. 114 peptide129 AKGEAAAERSEQ ID NO. 129 peptide145 AKGEAAAE SEQ ID NO. 145 peptide162 AKGEAAASEQ ID NO. 162 peptide180 AKGEAA SEQ ID NO. 180 peptide199 AKGEASEQ ID NO. 199 peptide219 AKGE  SEQ ID NO. 219 peptide55 KGEAAAERPGEAAVASEQ ID NO. 55 peptide65 KGEAAAERPGEAAV SEQ ID NO. 65 peptide76KGEAAAERPGEAA SEQ ID NO. 76 peptide88 KGEAAAERPGEA SEQ ID NO. 88peptide101 KGEAAAERPGE SEQ ID NO. 101 peptide115 KGEAAAERPGSEQ ID NO. 115 peptide130 KGEAAAERP SEQ ID NO. 130 peptide146 KGEAAAER SEQ ID NO. 146 peptide163 KGEAAAE  SEQ ID NO. 163 peptide181 KGEAAA SEQ ID NO. 181 peptide200 KGEAA  SEQ ID NO. 200 peptide220 KGEA SEQ ID NO. 220 peptide66 GEAAAERPGEAAVA SEQ ID NO. 66 peptide77GEAAAERPGEAAV SEQ ID NO. 77 peptide89 GEAAAERPGEAA SEQ ID NO. 89peptide102 GEAAAERPGEA SEQ ID NO. 102 peptide116 GEAAAERPGESEQ ID NO. 116 peptide131 GEAAAERPG SEQ ID NO. 131 peptide147 GEAAAERP SEQ ID NO. 147 peptide164 GEAAAER  SEQ ID NO. 164 peptide182 GEAAAE SEQ ID NO. 182 peptide201 GEAAA  SEQ ID NO. 201 peptide221 GEAA SEQ ID NO. 221 peptide78 EAAAERPGEAAVA SEQ ID NO. 78 peptide90EAAAERPGEAAV SEQ ID NO. 90 peptide103 EAAAERPGEAA SEQ ID NO. 103peptide117 EAAAERPGEA SEQ ID NO. 117 peptide132 EAAAERPGE SEQ ID NO. 132peptide148 EAAAERPG  SEQ ID NO. 148 peptide165 EAAAERP  SEQ ID NO. 165peptide183 EAAAER  SEQ ID NO. 183 peptide202 EAAAE  SEQ ID NO. 202peptide222 EAAA  SEQ ID NO. 222 peptide91 AAAERPGEAAVA SEQ ID NO. 91peptide104 AAAERPGEAAV SEQ ID NO. 104 peptide118 AAAERPGEAASEQ ID NO. 118 peptide133 AAAERPGEA SEQ ID NO. 133 peptide149 AAAERPGE SEQ ID NO. 149 peptide166 AAAERPG  SEQ ID NO. 166 peptide184 AAAERP SEQ ID NO. 184 peptide203 AAAER  SEQ ID NO. 203 peptide223 AAAE SEQ ID NO. 223 peptide105 AAERPGEAAVA SEQ ID NO. 105 peptide119AAERPGEAAV SEQ ID NO. 119 peptide134 AAERPGEAA SEQ ID NO. 134 peptide150AAERPGEA  SEQ ID NO. 150 peptide167 AAERPGE  SEQ ID NO. 167 peptide185AAERPG  SEQ ID NO. 185 peptide204 AAERP  SEQ ID NO. 204 peptide224 AAER SEQ ID NO. 224 peptide120 AERPGEAAVA SEQ ID NO. 120 peptide135 AERPGEAAVSEQ ID NO. 135 peptide151 AERPGEAA  SEQ ID NO. 151 peptide168 AERPGEA SEQ ID NO. 168 peptide186 AERPGE  SEQ ID NO. 186 peptide205 AERPG SEQ ID NO. 205 peptide225 AERP  SEQ ID NO. 225 peptide136 ERPGEAAVASEQ ID NO. 136 peptide152 ERPGEAAV  SEQ ID NO. 152 peptide169 ERPGEAA SEQ ID NO. 169 peptide187 ERPGEA  SEQ ID NO. 187 peptide206 ERPGE SEQ ID NO. 206 peptide226 ERPG  SEQ ID NO. 226 peptide153 RPGEAAVA SEQ ID NO. 153 peptide170 RPGEAAV  SEQ ID NO. 170 peptide188 RPGEAA SEQ ID NO. 188 peptide207 RPGEA  SEQ ID NO. 207 peptide227 RPGE SEQ ID NO. 227 peptide171 PGEAAVA  SEQ ID NO. 171 peptide189 PGEAAV SEQ ID NO. 189 peptide208 PGEAA  SEQ ID NO. 208 peptide228 PGEA SEQ ID NO. 228 peptide190 GEAAVA  SEQ ID NO. 190 peptide209 GEAAV SEQ ID NO. 209 peptide229 GEAA  SEQ ID NO. 229 peptide210 EAAVASEQ ID NO. 210 peptide230 EAAV  SEQ ID NO. 230 peptide231 AAVA SEQ ID NO. 231

As illustrated in FIG. 5, MARCKS was phosphorylated by PKC andconsequently translocated from the membrane to the cytoplasm. Here, PKGappeared to induce dephosphorylation of MARCKS (FIG. 2A, lane 4, andFIG. 2B). This dephosphorylation was reversed by the PKG inhibitorR_(p)-8-Br-PET-cGMP (FIG. 2A, lane 5), indicating the dephosphorylationwas specifically PKG-dependent. In FIG. 2, the NHBE cells were labeledwith [³²P]orthophosphate and then exposed to the indicated reagents.MARCKS phosphorylation in response to the treatments was evaluated byimmunoprecipitation assay. In FIG. 2A, 8-Br-cGMP reversed MARCKSphosphorylation induced by PMA, and this effect of 8-Br-cGMP could beblocked by R_(p)-8-Br-PET-cGMP (PKG inhibitor) or okadaic acid (PP1/2Ainhibitor). For FIG. 2B, PMA-induced phosphorylation of MARCKS wasreversed by subsequent exposure of cells to 8-Br-cGMP. Lane 1, mediumalone for 8 min; lane 2, 100 nM PMA for 3 min; lane 3, 100 nM PMA for 3min and then with 1 μM 8-Br-cGMP for 5 min; lane 4, 100 nM PMA for 8min; lane 5, medium alone for 3 min and then 100 nM PMA+1 μM 8-Br-cGMPfor 5 min. In FIG. 2C, 8-Br-cGMP-induced MARCKS dephosphorylation wasattenuated by fostriecin in a concentration-dependent manner.

It is believed that PKG acts to dephosphorylate MARCKS via activation ofa protein phosphatase. As illustrated in FIG. 2A (lane 6), okadaic acidat 500 nM, a concentration that could inhibit both PP1 and PP2A, blockedPKG-induced dephosphorylation of MARCKS, suggesting that PKG causeddephosphorylation by activating PP1 and/or PP2A. Further studies withfostriecin and direct assay of phosphatase activities indicated thatonly PP2A was activated by PKG and was responsible for removal of thephosphate groups from MARCKS (FIG. 2C). It is likely that either okadaicacid or fostriecin, at concentrations that inhibited PKG-induceddephosphorylation of MARCKS, attenuated mucin secretion induced byPMA+8-Br-cGMP or UTP as exhibited in FIG. 3. FIG. 3 helps to demonstratethat PP2A is an essential component of the mucin secretory pathway. NHBEcells were preincubated with the indicated concentration of fostriecin,okadaic acid (500 nM), or medium alone for 15 min and then stimulatedwith PMA (100 nM)+8-Br-cGMP (1 μM) for 15 min or with UTP (100 μM) for 2h. Secreted mucin was measured by ELISA. Data are presented asmean.+−.S.E. (n=6 at each point) wherein * stands for significantlydifferent from medium control (p<0.05); † stands for significantlydifferent from PMA+8-Br-cGMP stimulation (p<0.05); and ‡ stands forsignificantly different from UTP stimulation p<0.05). Thus,dephosphorylation of MARCKS by a PKG-activated PP2A appears to be anessential component of the signaling pathway leading to mucin granuleexocytosis.

To reveal molecular events by which MARCKS links kinase activation tomucin secretion, phosphorylation of MARCKS in response to PKC/PKGactivation was investigated in depth. As illustrated in FIG. 1A, PMA(100 nM) likely induced a significant increase (3-4-fold) in MARCKSphosphorylation in NHBE cells, and this phosphorylation was attenuatedby the PKC inhibitor calphostin C (500 nM). Once phosphorylated, MARCKSwas translocated from the plasma membrane to the cytoplasm (FIG. 1B).More specifically, FIG. 1A shows the activation of PKC results in MARCKSphosphorylation in NHBE cells. Cells were labeled with[³²P]orthophosphate for 2 h and then exposed to the stimulatory and/orinhibitory reagents. MARCKS phosphorylation in response to thetreatments was evaluated by immunoprecipitation as described. Lane 1,medium control; lane 2 the vehicle, 0.1% Me.sub.2SO; lane 3, 100 nM4α-PMA; lane 4, 100 nM PMA; lane 5, 100 nM PMA+500 nM calphostin C; lane6, 500 nM calphostin C. FIG. 1B demonstrates phosphorylated MARCKS istranslocated from the plasma membrane to the cytoplasm. ³²P-Labeledcells were exposed to PMA (100 nM) or medium alone for 5 min, and thenthe membrane and the cytosol fractions were isolated. Activation of PKGby 8-Br-cGMP (1 μM, another kinase activation event necessary forprovoking mucin secretion, did not lead to MARCKS phosphorylation, but,in fact, the opposite effect was observed: MARCKS phosphorylationinduced by PMA was reversed by 8-Br-cGMP (FIG. 2A). This effect of8-Br-cGMP was not due to suppression of PKC activity, as the PMA-inducedphosphorylation could be reversed by subsequent addition of 8-Br-cGMP tothe cells (FIG. 2B). Therefore, PKG activation likely results indephosphorylation of MARCKS.

Further investigation demonstrated that PKG-induced MARCKSdephosphorylation was blocked by 500 nM okadaic acid, a proteinphosphatase (type 1 and/or 2A (PP1/2A)) inhibitor (FIG. 2A, lane 6).Thus, it appeared that the dephosphorylation was mediated by PP1 and/orPP2A. To define the subtype of protein phosphatase involved, a novel andmore specific inhibitor of PP2A, fostriecin (IC₅₀=3.2 nM), was utilizedin additional phosphorylation studies. As illustrated in FIG. 2C,fostriecin inhibited PKG-induced MARCKS dephosphorylation in aconcentration-dependent manner (1-500 nM), suggesting that PKG inducedthe dephosphorylation via activation of PP2A. To confirm furtheractivation of PP2A by PKG in NHBE cells, cytosolic PP1 and PP2Aactivities were determined after exposure of the cells to 8-Br-cGMP.PP2A activity was increased approximately 3-fold (from 0.1 to 0.3nmol/min/mg proteins, p<0.01) at concentrations of 8-Br-cGMP as low as0.1 .mu.M, whereas PP1 activity remained unchanged. This data indicatesthat PP2A may be activated by PKG and is responsible for thedephosphorylation of MARCKS. Accordingly, this PP2A activity appearedcritical for mucin secretion to occur; when PKG-induced MARCKSdephosphorylation was blocked by okadaic acid or fostriecin, thesecretory response to PKC/PKG activation or UTP stimulation wasameliorated (FIG. 3).

MARCKS Associates with Actin and Myosin in the Cytoplasm

FIG. 4 depicts a radiolabeled immunoprecipitation assay which revealsthat MARCKS may associate with two other proteins (about 200 and about40 kDa) in the cytoplasm. In FIG. 4 NHBE cells were labeled with[³H]leucine and [³H]proline overnight, and the membrane and the cytosolfractions were prepared as described under “Experimental Procedures.”Isolated fractions were precleared with the nonimmune control antibody(6F6). The cytosol was then divided equally into two fractions and usedfor immunoprecipitation carried out in the presence of 10 μMcytochalasin D (Biomol, Plymouth Meeting, Pa.) with the anti-MARCKSantibody 2F12 (lane 2) and the nonimmune control antibody 6F6 (lane 3),respectively. MARCKS protein in the membrane fraction was also assessedby immunoprecipitation using the antibody 2F12 (lane 1). Theprecipitated protein complex was resolved by 8% SDS-polyacrylamide gelelectrophoresis and visualized by enhanced autoradiography. MARCKSappeared to associate with two cytoplasmic proteins with molecularmasses of about 200 and about 40 kDa, respectively. These twoMARCKS-associated proteins were excised from the gel and analyzed bymatrix-assisted laser desorption ionization/time of flight massspectrometry/internal sequencing (the Protein/DNA Technology Center ofRockefeller University, N.Y.). The obtained peptide mass and sequencedata were used to search protein databases via Internet programsProFound and MS-Fit. Results indicate that they are myosin (heavy chain,non-muscle type A) and actin, respectively. Matrix-assisted laserdesorption ionization/time of flight mass spectrometry/internal sequenceanalysis indicates that these two MARCKS-associated proteins were myosin(heavy chain, non-muscle type A) and actin, respectively.

These studies suggest a new paradigm for the signaling mechanismcontrolling exocytotic secretion of airway mucin granules as well asproviding what is believed to be the first direct evidence demonstratinga specific biological function of MARCKS in a physiological process.MARCKS serves as a key mediator molecule regulating mucin granulerelease in human airway epithelial cells. It is believed thatelicitation of airway mucin secretion requires dual activation andsynergistic actions of PKC and PKG. Activated PKC phosphorylates MARCKS,resulting in translocation of MARCKS from the inner face of the plasmamembrane into the cytoplasm. Activation of PKG in turn activates PP2A,which dephosphorylates MARCKS in the cytoplasm. Because the membraneassociation ability of MARCKS is dependent on its phosphorylation statethis dephosphorylation may allow MARCKS to regain its membrane-bindingcapability and may enable MARCKS to attach to membranes of cytoplasmicmucin granules. By also interacting with actin and myosin in thecytoplasm (FIG. 4), MARCKS may then be able to tether granules to thecellular contractile apparatus, mediating granule movement to the cellperiphery and subsequent exocytotic release. The wide distribution ofMARCKS suggests the possibility that this or a similar mechanism mayregulate secretion of membrane-bound granules in various cell typesunder normal or pathological conditions.

As indicated in FIG. 5, MARCKS may function as a molecular linker byinteracting with granule membranes at its N-terminal domain and bindingto actin filaments at its PSD site, thereby tethering granules to thecontractile cytoskeleton for movement and exocytosis. FIG. 5 shows apossible mechanism depicting that mucin secretagogue interacts withairway epithelial (goblet) cells and activates two separate proteinkinases, PKC and PKG. Activated PKC phosphorylates MARCKS, causingMARCKS translocation from the plasma membrane to the cytoplasm, whereasPKG, activated via the nitric oxide (NO)→GC-S→cGMP→PKG pathway, in turnactivates a cytoplasmic PP2A, which dephosphorylates MARCKS. Thisdephosphorylation stabilizes MARCKS attachment to the granule membranes.In addition, MARCKS also interacts with actin and myosin, therebylinking granules to the cellular contractile machinery for subsequentmovement and exocytotic release of inflammatory mediators, such as MPO.The attachment of MARCKS to the granules after it is released into thecytoplasm may also be guided by specific targeting proteins or someother forms of protein-protein interactions in which the N-terminaldomain of MARCKS is involved. In either case, the MANS peptide, or anactive fragment thereof, comprising at least 4 amino acids, would act toinhibit competitively targeting of MARCKS to the membranes of mucingranules, thereby blocking secretion.

The invention also relates to a new method for blocking any cellularexocytotic secretory process, especially those releasing inflammatorymediators from granules contained within inflammatory cells, whosestimulatory pathways involve the protein kinase C (PKC) substrate MARCKSprotein and release of contents from membrane-bound vesicles.Specifically, the inventors have shown that stimulated release of theinflammatory mediator myloperoxidase from human (FIG. 6) or canine (FIG.7) neutrophils can be blocked in a concentration-dependent manner by theMANS peptide. Specifically, FIG. 6 shows isolated neutrophils that werestimulated to secrete myloperoxidase (MPO) with 100 nM PMA and 10 .mu.M8-Br-cGMP. 100 μM MANS peptide decreased secretion of MPO to controllevels (*=p<0.05). 10 μM MANS causes a slight decrease in MPO secretion.10 or 100 μM of a control peptide (RNS) has no effect on MPO secretion.In FIG. 7, isolated neutrophils were stimulated to secretemyloperoxidase (MPO) with 100 nM PMA and 10 μM 8-Br-cGMP. 100 μM MANSpeptide decreased secretion of MPO to control levels (*=p<0.05). 10 μMMANS causes a slight decrease in MPO secretion. 10 or 100 μM of acontrol peptide (RNS) has no effect on MPO secretion. Thus, the peptidemay be used therapeutically to block the release of mediators ofinflammation secreted from infiltrating inflammatory cells in anytissues. Many of these released mediators are responsible for theextensive tissue damage observed in a variety of chronic inflammatorydiseases (i.e., respiratory diseases such as asthma, chronic bronchitisand COPD, inflammatory bowel diseases including ulcerative colitis andCrohn's disease, autoimmune diseases, skin diseases such as rosacea,eczema; and severe acne, arthritic and pain syndromes such as rheumatoidarthritis and fibromyalgia). This invention may be useful for treatingdiseases such as arthritis, chronic bronchitis, COPD and cysticfibrosis. This invention is accordingly useful for the treatment in bothhuman and animal diseases, especially those affecting equines, canines,felines, and other household pets.

FIGS. 8-12 show MPO secretion for both humans and canines. In all ofthese experiments, isolated neutrophils were stimulated with LPS at aconcentration of 1×10⁻⁶ M for 10 minutes at 37° C. prior to adding thestimuli as indicated in the figures. The LPS primes the cells so theycan respond to a secretagogue.

In one embodiment, this invention discloses a method of regulating aninflammation in a subject comprising administering a therapeuticallyeffective amount of a pharmaceutical composition comprising a MANSpeptide or an active fragment thereof. In one aspect of this embodiment,said active fragment of the MANS protein comprises at least four andpreferably six amino acids. In another aspect, said inflammation iscaused by respiratory diseases, bowel diseases, skin diseases,autoimmune diseases and pain syndromes. In another aspect, saidrespiratory diseases are selected from the group consisting of asthma,chronic bronchitis, and COPD. In another aspect, said bowel diseases areselected from the group consisting of ulcerative colitis, Crohn'sdisease and irritable bowel syndrome. In another aspect, said skindiseases are selected from the group consisting of rosacea, eczema,psoriasis and severe acne. In another aspect, said inflammation iscaused by arthritis or cystic fibrosis. In another aspect, said subjectis a mammal. Additionally, in another aspect, said mammal is selectedfrom the group consisting of humans, canines, equines and felines. Inanother aspect, said administering step is selected from the groupconsisting of topical administration, parenteral administration, rectaladministration, pulmonary administration, nasal administration,inhalation and oral administration. In another aspect, said pulmonaryadministration is selected from the group of aerosol, dry powderinhaler, metered dose inhaler, and nebulizer.

In another embodiment, this invention discloses a method for regulatinga cellular secretory process in a subject comprising administering atherapeutically effective amount of a pharmaceutical compositioncomprising at least one compound comprising a MANS peptide or an activefragment thereof, that regulates an inflammatory mediator in a subject.In one aspect of this embodiment, said active fragment of the MANSprotein comprises at least four, and preferably six amino acids. Inanother aspect, said regulating a cellular secretory process is blockingor reducing a cellular secretory process. In another aspect, saidinflammatory mediator is caused by respiratory diseases, bowel diseases,skin diseases, autoimmune diseases and pain syndromes. In anotheraspect, said respiratory diseases are selected from the group consistingof asthma, chronic bronchitis, and COPD. In another aspect, said boweldiseases are selected from the group consisting of ulcerative colitis,Crohn's disease and irritable bowel syndrome. In another aspect, saidskin diseases are selected from the group consisting of rosacea, eczema,psoriasis and severe acne. In another aspect, said inflammatory mediatoris caused by arthritis or cystic fibrosis. In another aspect, saidsubject is a mammal. In another aspect, said mammal is selected from thegroup consisting of humans, canines, equines and felines. In anotheraspect, said administering step is selected from the group consisting oftopical administration, parenteral administration, rectaladministration, pulmonary administration, nasal administration,inhalation and oral administration. In another aspect, said pulmonaryadministration is selected from the group of aerosol, dry powderinhaler, metered dose inhaler, and nebulizer.

In another embodiment, this invention discloses a method of reducinginflammation in a subject comprising administering a therapeuticallyeffective amount of a compound that inhibits the MARCKS-related releaseof inflammatory mediators, whereby the release of inflammatory mediatorsin the subject is reduced compared to that which would occur in theabsence of said treatment. In one aspect of this embodiment, saidcompound is at least one active fragment of a MARCKS protein. In anotheraspect, said active fragment is at least four and preferably six aminoacids in length. In another aspect, said compound is a MANS peptide oran active fragment thereof. In another aspect, said compound is anantisense oligonucleotide directed against the coding sequence of aMARCKS protein or an active fragment thereof. In another aspect, saidactive fragment is at least four and preferably six amino acids inlength.

In another embodiment, this invention discloses a method of reducinginflammation in a subject comprising administering a therapeuticallyeffective amount of a pharmaceutically active composition comprising acompound that inhibits the MARCKS-related release of inflammatorymediators, whereby the inflammation in the subject is reduced comparedto that which would occur in the absence of said treatment. In oneaspect of this embodiment, said compound is an active fragment of aMARCKS protein. In another aspect, said active fragment is at least fourand preferably six amino acids in length. In another aspect, saidcompound is a MANS peptide or an active fragment thereof. In anotheraspect, said compound is an antisense oligonucleotide directed againstthe coding sequence of a MARCKS protein or an active fragment thereof.In another aspect, said active fragment is at least four and preferablysix amino acids in length. The present invention is intended toencompass a composition that contains one or more of the MANS peptide orits active fragments and use thereof in the treatment of inhibiting therelease of inflammatory mediators from granules or vesicles ofinflammatory cells.

In another embodiment, this invention discloses a method of reducing orinhibiting inflammation in a subject comprising administering atherapeutically effective amount of at least one peptide comprising MANSpeptide or an active fragment thereof effective to inhibit or suppressrelease of an inflammatory mediator at the inflammation site. In oneaspect of this embodiment, said active fragment is at least four andpreferably at least six amino acids in length. In another aspect, saidinflammatory mediators are produced by cells selected from the groupconsisting of neutrophils, basophils, eosinophils, monocytes andleukocytes. Preferably the cells are leukocytes, more preferablygranulocytes, and even more preferably neutrophils, basophils,eosinophils or a combination thereof. In another aspect, the agent isadministered orally, parenterally, cavitarily, rectally or through anair passage. In another aspect, said composition further comprises asecond molecule selected from the group consisting of an antibiotic, anantiviral compound, an antiparasitic compound, an anti-inflammatorycompound, and an immunosuppressant.

An active fragment of a MANS peptide can be selected from the groupconsisting of the peptides of disclosed in Table 1. As disclosed herein,these peptides may be contain optional chemical moieties at theN-terminal and/or C-terminal amino acid.

In another aspect of this invention, the methods disclosed in thisinvention can be accomplished by use of or administering of combinationsof the peptides disclosed in the invention in Table 1, i.e., by use ofor administering of one or more of these peptides. Preferably a singlepeptide is used or administered in the methods disclosed herein.

In response to protein kinase C (PKC) activation by an inflammatorystimulant, degranulation in a cell selected from the group consisting ofneutrophils, eosinophils, monocytes/macrophages and lymphocytes can beattenuated by pre-incubation and by co-incubation with a peptideidentical to the N-terminal region of MARCKS protein, wherein thepeptide is selected from the group of MANS peptide fragments asdisclosed in Table 1. Although time courses and concentrations can varyamong cell types, in all cases the MANS peptide attenuates PKC-induceddegranulation.

Having now described the invention, the same will be illustrated withreference to certain examples, which are included herein forillustration purposes only, and which are not intended to be limiting ofthe invention.

EXAMPLES Methods and Materials

Radiolabeled Immunoprecipitation Assay

When labeling with [³²P]phosphate, cells were preincubated for 2 h inphosphate-free Dulbecco's modified Eagle's medium containing 0.2% bovineserum albumin and then labeled with 0.1 mCi/ml [³²P]orthophosphate (9000Ci/mmol, PerkinElmer Life Sciences) for 2 h. For labeling with[³H]myristic acid or ³H-amino acids, cells were incubated overnight inmedium containing 50 μCi/ml [³H]myristic acid (49 Ci/mmol, PerkinElmerLife Sciences) or 0.2 mCi/ml [³H]leucine (159 Ci/mmol, PerkinElmer LifeSciences) plus 0.4 mCi/ml [³H]proline (100 Ci/mmol, PerkinElmer LifeSciences). Following labeling, cells were exposed to stimulatoryreagents for 5 min. When an inhibitor was used, cells were preincubatedwith the inhibitor for 15 min prior to stimulation. At the end of thetreatments, cells were lysed in a buffer containing 50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Nonidet P-40, 1 mMphenylmethylsulfonyl fluoride, 1 mM benzamidine, 10 μg/ml pepstatin A,and 10 μg/ml leupeptin. Trichloroacetic acid precipitation andscintillation counting may determine the radiolabeling efficiency ineach culture. Immunoprecipitation of MARCKS protein was carried outaccording to the method of Spizz and Blackshear using cell lysatescontaining equal counts/min. Spizz et al., J. Biol. Chem. 271, 553-562(1996). Precipitated proteins were resolved by 8% SDS-polyacrylamide gelelectrophoresis and visualized by autoradiography. Anti-human MARCKSantibody (2F12) and nonimmune control antibody (6F6) were used in thisassay.

To assess MARCKS or MARCKS-associated protein complexes in differentsubcellular fractions, radiolabeled and treated cells were scraped intoa homogenization buffer (50 mM Tris-HCl (pH 7.5), 10 mM NaCl, 1 mM EDTA,1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 10 μg/ml pepstatinA, 10 μg/ml leupeptin) and then disrupted by nitrogen cavitation (800pounds/square inch for 20 min at 4° C.). Cell lysates were centrifugedat 600×g for 10 min at 4° C. to remove nuclei and unbroken cells.Post-nuclear supernatants were separated into membrane and cytosolfractions by ultracentrifugation at 400,000×g for 30 min at 4° C. Themembrane pellet was solubilized in the lysis buffer by sonication.Immunoprecipitation was then carried out as described above.

MARCKS-Related Peptides

Both the myristoylated N-terminal sequence (MANS) and the randomN-terminal sequence (RNS) peptides were synthesized at GenemedSynthesis, Inc. (San Francisco, Calif.), then purified by high pressureliquid chromatography (>95% pure), and confirmed by mass spectroscopywith each showing one single peak with an appropriate molecular mass.The MANS peptide consisted of sequence identical to the first 24 aminoacids of MARCKS, i.e. the myristoylated N-terminal region that mediatesMARCKS insertion into membranes, MA-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1 (where MA is N-terminal myristoyl chain). The corresponding controlpeptide (RNS) contained the same amino acid composition as the MANS butarranged in random order, MA-GTAPAAEGAGAEVKRASAEAKQAF (SEQ ID NO: 232).The presence of the hydrophobic myristate moiety in these syntheticpeptides enhances their permeability to the plasma membranes, enablingthe peptides to be taken up readily by cells. To determine the effectsof these peptides on mucin secretion, cells were preincubated with thepeptides for 15 min prior to addition of secretagogues, and mucinsecretion was then measured by ELISA.

Antisense Oligonucleotides

MARCKS antisense oligonucleotide and its corresponding controloligonucleotide were synthesized at Biognostik GmbH (Gottingen,Germany). NHBE cells were treated with 5 μM antisense or controloligonucleotide apically for 3 days (in the presence of 2 μg/mllipofectin for the first 24 h). Cells were then incubated withsecretagogues, and mucin secretion was measured by ELISA. Total RNA andprotein were isolated from treated cells. MARCKS mRNA was assessed byNorthern hybridization according to conventional procedures using humanMARCKS cDNA as a probe. MARCKS protein level was determined by Westernblot using purified anti-MARCKS IgG1 (clone 2F12) as the primarydetection antibody.

Transient Transfection

The phosphorylation site domain (PSD) of MARCKS contains thePKC-dependent phosphorylation sites and the actin filament-binding site.To construct a PSD-deleted MARCKS cDNA, two fragments flanking the PSDsequence (coding for 25 amino acids) were generated by polymerase chainreaction and then ligated through the XhoI site that was attached to the5′-ends of oligonucleotide primers designed for the polymerase chainreaction. The resultant mutant cDNA and the wild-type MARCKS cDNA wereeach inserted into a mammalian expression vector pcDNA4/TO (Invitrogen,Carlsbad, Calif.). Isolated recombinant constructs were confirmed byrestriction digests and DNA sequencing.

HBE1 is a papilloma virus-transformed human bronchial epithelial cellline capable of mucin secretion when cultured in air/liquid interface.Transfection of HBE1 cells was carried out using the Effectenetransfection reagent (Qiagen, Valencia, Calif.) according to themanufacturer's instructions. Briefly, differentiated HBE1 cells grown inair/liquid interface were dissociated by trypsin/EDTA and re-seeded in12-well culture plates at 1×10⁵ cells/cm². After overnight incubation,cells were transfected with the wild-type MARCKS cDNA, the PSD-truncatedMARCKS cDNA, or vector DNA. Cells were cultured for 48 h to allow geneexpression and then exposed to secretagogues and mucin secretionmeasured by ELISA. All transfections were carried out in the presence ofpcDNA4/TO/lacZ plasmid (Invitrogen) (DNA ratio 6:1, total 1 μg DNA,ratio of DNA to Effectene reagent=1:25) to monitor variations intransfection efficiency. Results showed no significant difference in.beta.-galactosidase activities in cell lysates isolated from thetransfected cells, indicating similar transfection efficiency amongdifferent DNA constructs (data not shown).

Protein Phosphatase Activity Assay

PP1 and PP2A activities were measured using a protein phosphatase assaysystem (Life Technologies, Inc.) as known in the art with slightmodification. Huang et al., Adv. Exp. Med Biol. 396, 209-215 (1996).Briefly, NHBE cells were treated with 8-Br-cGMP or medium alone for 5min. Cells were then scraped into a lysis buffer (50 mM Tris-HCl (pH7.4), 0.1% .beta.-mecaptoethanol, 0.1 mM EDTA, 1 mM benaamidine, 10μg/ml pepstatin A, 10 μg/ml leupeptin) and disrupted by sonication for20 s at 4° C. Cell lysates were centrifuged and the supernatants savedfor phosphatase activity assay. The assay was performed using³²P-labeled phosphorylase A as a substrate. Released ³²P_(i) was countedby scintillation. The protein concentration of each sample wasdetermined by the Bradford assay. PP2A activity was expressed as thesample total phosphatase activity minus the activity remaining in thepresence of 1 nM okadaic acid. PP1 activity was expressed as thedifference between the activities remaining in the presence of 1 nM and1 μM okadaic acid, respectively. Protein phosphatase activities werereported as nmol of P_(i) released per min/mg total protein.

Cytotoxicity Assay

All reagents used in treating NHBE cells were examined for cytotoxicityby measuring the total release of lactate dehydrogenase from the cells.The assay was carried out using the Promega Cytotox 96 Kit according tothe manufacturer's instructions. All experiments were performed withreagents at non-cytotoxic concentrations.

Statistical Analysis

Data were analyzed for significance using one-way analysis of variancewith Bonferroni post-test corrections. Differences between treatmentswere considered significant at p<0.05.

Isolation of PMNs from Canine Blood

The steps involved in isolating PMN include collecting 10 ml ACDanticoagulated blood. Then layering 5 ml on 3.5 ml PMN isolation mediawhile ensuring that the PMN isolation media (IM) was at room temperature(RI). Next, the blood was centrifuged at room temperature for 30′, 550×gat 1700 RPMs. The low lower white band was transferred into 15 mlconical centrifuge tube (CCFT). Next, 2V HESS with 10% fetal bovineserum (PBS) was added and centrifuged at room temperature for 10′, 400×gat 1400 RPMs. The pellet was then resuspended in 5 ml 1-1ESS with PBS.The cell suspension was added to 50 ml CCFT containing 20 ml of ice cold0.88% NH₄Cl and inverted two to three times. The resulting product wascentrifuged for 10′, 800×g at 2000 RPMs, then aspirated and resuspendedin 5 ml HBSS with FBS. The prep was examined by counting and cytospinand preferably for whole blood, the cell number should be between10⁹-10¹¹ cells and for PMNs, cell number should be between 2-4×10⁷cells. See generally, Wang et al., J. Immunol., “Neutrophil-inducedchanges in the biomechanical properties of endothelial cells: roles ofICAM-1 and reactive oxygen species,” 6487-94 (2000).

MPO Colorimetric Enzyme Assay

Samples were assayed for MPO activity in 96 well round bottom microtiterplates using a sandwich ELISA kit (R & D Systems, Minneapolis, Minn.).Briefly, 20 microliters of sample is mixed with 180 microliters ofsubstrate mixture containing 33 mM potassium phosphate, pH 6.0, 0.56%Triton X-100, 0.11 mM hydrogen peroxide, and 0.36 mM O-DiannisidineDihydrochloride in an individual microtiter well. The finalconcentrations in the assay mixture are: 30 mM potassium phosphate, pH6.0, 0.05% Triton X-100, 0.1 mM hydrogen peroxide, and 0.32 mM0-Diannisidine Dihydrochloride. After mixing, the assay mixture wasincubated at room temperature for 5 minutes, and MPO enzyme activitydetermined spectrophotometrically at 550 nanometers. Samples wereassayed in duplicate.

Example 1 Inflammatory Mediator Secretion Studies

Four different leukocyte types or models that secrete specific granulecontents in response to phorbol ester induced activation of PKC wereused. Neutrophils were isolated from human blood and in vitro release ofMPO by these cells was assessed. Release of membrane-bound inflammatorymediators from commercially-available human leukocyte cell lines wasalso evaluated. The human promyelocytic cell line HL-60 clone 15 wasused to assess secretion of EPO (Fischkoff S A. Graded increase inprobability of eosinophilic differentiation of HL-60 promyelocyticleukemia cells induced by culture under alkaline conditions. Leuk Res1988; 12:679-686; Rosenberg H F, Ackerman S J, Tenen D G. Humaneosinophil cationic protein: molecular cloning of a cytotoxin andhelminthotoxin with ribonuclease activity. J Exp Med 1989; 170:163-176;Tiffany H L, Li F, Rosenberg H F. Hyperglycosylation of eosinophilribonucleases in a promyelocytic leukemia cell line and indifferentiated peripheral blood progenitor cells. J Leukoc Biol 1995;58:49-54; Badewa A P, Hudson C E, Heiman A S. Regulatory effects ofeotaxin, eotaxin-2, and eotaxin-3 on eosinophil degranulation andsuperoxide anion generation. Exp Biol Med 2002; 227:645-651). Themonocytic leukemia cell line U937 was used to assess secretion oflysozyme (Hoff T, Spencker T, Emmendoerffer A., Goppelt-Struebe M.Effects of glucocorticoids on the TPA-induced monocytic differentiation.J Leukoc Biol 1992; 52:173-182; Balboa M A, Saez Y, Balsinde J.Calcium-independent phospholipase A2 is required for lysozyme secretionin U937 promonocytes. J Immunol 2003; 170:5276-5280; Sundstrom C,Nilsson K. Establishment and characterization of a human histiocyticlymphoma cell line (U-937). Int J Cancer 1976; 17:565-577). Thelymphocyte natural killer cell line NK-92 used to assess release ofgranzyme (Gong J H., Maki G, Klingemann H G. Characterization of a humancell line (NK-92) with phenotypical and functional characteristics ofactivated natural killer cells. Leukemia 1994; 8:652-658; Maki G,Klingemann H G, Martinson J A, Tam Y K. Factors regulating the cytotoxicactivity of the human natural killer cell line, NK-92. J Hematother StemCell Res 2001; 10:369-383; Takayama H, Trenn G, Sitkovsky M V. A novelcytotoxic T lymphocyte activation assay. J Immunol Methods 1987;104:183-190). In all cases, the cells were preincubated with a range ofconcentrations of a synthetic peptide identical to the 24 amino acidMARCKS N-terminus (MANS—myristoylated N-terminal sequence peptide;MA-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1) wherein MA is myristoylattached to the N-terminal amine of the peptide by an amide bond), or amissense control peptide (RNS: Random N-terminal sequence peptide;MA-GTAPAAEGAGAEVKRASAEAKQAF, SEQ ID NO: 232) which consists of the same24 amino acids but arranged in random order sequence which possessesless than 13% sequence identity to the MANS peptide sequence.Alternatively, the cells were pretreated with one of the synthetictruncated peptides listed in Table 3 below.

In each of the cell types, MANS, but not RNS, attenuates release ofinflammatory mediators in a concentration-dependent manner. A usefultime course of observation is 0.5-3.0 hrs. The results are consistentwith the N-terminal region of the MARCKS protein being involved inintracellular pathways leading to leukocyte degranulation.

Human Neutrophil Isolation

These studies were approved by the human studies Institutional ReviewBoard (IRB). Human neutrophils were isolated as previously described(see Takashi S, OkuboY, Horie S. Contribution of CD54 to humaneosinophil and neutrophil superoxide production. J Appl Physiol 2001;91:613-622) with slight modifications. Briefly, heparinized venous bloodwas obtained from normal healthy volunteers, diluted with RPMI-1640(Cellgro; Mediatech, Inc., Herndon, Va.) at a ratio of 1:1, layered ontoa Histopaque (density, 1.077 g/ml; Sigma-Aldrich Co., St. Louis, Mo.)and centrifuged at 400 g for 20 min at 4° C. The supernatant andmononuclear cells at the interface were carefully removed, anderythrocytes in the sediment were lysed in chilled distilled water.Isolated granulocytes were washed twice with Hanks' balanced saltssolution (HBSS) and resuspended in HBSS on ice. The neutrophils used forthe experiments were of >98% purity with <2% contamination byeosinophils, and the viability was >99% as determined by Trypan blue dyeexclusion.

Measurement of Released Neutrophil MPO Activity

For measurement of MPO release, purified human neutrophils suspended inHBSS were aliquoted at 4×10⁶ cells/ml in 15 ml tubes and preincubatedwith either 50 or 100 μM of MANS, RNS, or one of the peptides ofinvention for 10 min at 37° C. The cells then were stimulated with 100nM phorbol 12-myristate 13-acetate (PMA) for up to 3 hrs. A controlreference (PMA control reference) was established using purified humanneutrophils suspended in HBSS aliquoted at 4×10⁶ cells/ml in 15 mL tubesand stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) in theabsence of a test peptide for the same time periods. The reaction wasterminated by placing the tubes on ice and centrifugation at 400 g for 5min at 4° C.

MPO activity in the cell supernatant was assayed usingtetramethylbenzidine (TMB) based on a previously established technique(Abdel-Latif D, Steward M, Macdonald D L, Francis G A., Dinauer M C,Lacy P. Rac2 is critical for neutrophil primary granule exocytosis.Blood 2004; 104:832-839). Briefly, 100 μL of TMB substrate solution wasadded to 50 μL of cell supernatants or standard human MPO (EMDBiosciences, Inc., San Diego, Calif.) in a 96-well microplate followedby incubation at room temperature for 15 min. The reaction wasterminated by addition of 50 μL of 1M H₂SO₄ and absorbance was read at450 nm in a spectrophotometric microplate reader (VERSA max, MolecularDevices, Sunnyvale, Calif.).

Leukocyte Culture Studies.

Three types of human leukocyte cell lines, specifically thepromyelocytic cell line HL-60 clone 15, the monocytic cell line U937,and the lymphocyte natural killer cell line NK-92 were purchased fromAmerican Type Culture Collection (ATCC; Rockville, Md.). HL-60 clone 15cells (ATCC CRL-1964) were maintained in medium consisting of RPMI 1640with L-glutamine supplemented with 10% heat-inactivated fetal bovineserum (Gibco; Invitrogen Co., Carlsbad, Calif.), 50 IU/ml penicillin, 50μg/mL streptomycin, and 25 mM HEPES buffer, pH 7.8, at 37° C. in anatmosphere containing 5% CO2. Final differentiation to aneosinophil-like phenotype was initiated by culturing cells at 5×10⁵cells/ml in the above medium containing 0.5 mM butyric acid(Sigma-Aldrich Co.) for 5 days as previously described (Tiffany H L, LiF, Rosenberg H F. Hyperglycosylation of eosinophil ribonucleases in apromyelocytic leukemia cell line and in differentiated peripheral bloodprogenitor cells. J Leukoc Biol 1995; 58:49-54; Tiffany H L, Alkhatib G,Combadiere C, Berger E A, Murphy P M. CC chemokine receptors 1 and 3 aredifferentially regulated by IL-5 during maturation of eosinophilic HL-60cells. J Immunol 1998; 160:1385-1392). U937 cells (ATCC CRL-1593.2) weregrown at 37° C. in an atmosphere of 5% CO₂ in complete medium consistingof RPMI 1640 with L-glutamine supplemented with 10% FBS, 50 IU/mlpenicillin, and 50 μg/mL streptomycin. NK-92 cells (ATCC CRL-2407) weremaintained in alpha-MEM medium (Sigma-Aldrich Co.) supplemented with 20%FBS, 100 U/ml of interleukin-2 (IL-2) (Chemicon International, Inc.,Temecula, Calif.), 5×10⁻⁵ M of 2-mercaptoethanol, 50 IU/mL penicillin,and 50 μg/ml streptomycin at 37° C. in an atmosphere containing 5% CO₂.Cell morphology was judged by assessment of Wright-Giemsa-stained cells.Viability of cells harvested for experiments was assessed by trypan blueexclusion and populations of cells with viability>95% were used.

Incubation of Cells for Degranulation Assays.

HL-60 clone 15, U937, and NK-92 cells were washed and resuspended at2.5×10⁶ cells/ml in phenol red-free RPMI-1640 (Cellgro; Mediatech, Inc.)for all degranulation assays. Aliquots of cells in 15 ml tubes werepreincubated with indicated concentrations of MANS, RNS, or a testpeptide for 10 min at 37° C. The cells then were stimulated with PMA forup to 2 hr. A control reference (PMA control reference) was establishedfor each cell type using HL-60 clone 15, U937, and NK-92 cells,respectively, which were washed and resuspended at 2.5×10⁶ cells/ml inphenol red-free RPMI-1640 and stimulated with PMA but in the absence ofMANS, RNS, or a test peptide for the same time periods. The reaction wasterminated by placing tubes on ice and centrifuging cells at 400 g for 5min at 4° C.

For measurements of released MPO from neutrophils and released lysozymefrom U937 cells, we were able to quantify secretion by using asstandards human MPO and egg white ovalbumin, respectively. For releasedEPO from HL-60 clone 15 cells and for released granzyme from NK-92cells, no standards were available to use for quantification. Hence,both released and intracellular (from lysed cells) levels of EPO andgranzyme were measured, and the released EPO and granzyme were expressedas a percentage of total (intracellular and released) for each. Tomeasure intracellular EPO in HL-60 clone 15 cells and intracellulargranzyme in NK-92 cells, appropriate aliquots of 0.1% triton X-100-lysedcells were taken for quantification of intracellular granule proteins asdescribed below. All treatments were expressed as percentage of controlto minimize variability between cultures.

Measurement of HL-60 EPO Release.

EPO activity released by HL-60 clone 15 cells was assayed using TMBaccording to a previously established technique (Lacy P, Mahmudi-Azer S,Bablitz B, Hagen S C, Velazquez J R, Man S F, Moqbel R. Rapidmobilization of intracellularly stored RANTES in response tointerferon-gamma in human eosinophils. Blood 1999; 94:23-32). Thus, 100μL of TMB substrate solution was added to 50 μL (μL=microliters) ofsample in a 96-well microplate and incubated at room temperature for 15min (min=minutes). The reaction was terminated by addition of 50 μL of1.0M H₂SO₄ and absorbance was read at 450 nm (nm=nanometers) in aspectrophotometric microplate reader. The amount of secreted EPO wasexpressed as percentage of total content, using the amount obtained inthe same number of triton X-100-lysed cells.

Measurement of Monocyte Lysozyme Secretion.

Lysozyme secreted by U937 cells was measured using a spectrophotometricassay as described previously (Balboa M A, Saez Y, Balsinde J.Calcium-independent phospholipase A2 is required for lysozyme secretionin U937 promonocytes. J Immunol 2003; 170:5276-5280) with slightmodification. Thus, 100 μL of sample was mixed with 100 μL of aMicrococcus lysodeikticus (Sigma-Aldrich Co.) suspension (0.3 mg/ml in0.1 M sodium phosphate buffer, pH 7.0) in a 96-well microplate. Thedecrease in absorbance at 450 nm was measured at room temperature. Acalibration curve was constructed using chicken egg white lysozyme (EMDBiosciences, Inc.) as a standard.

Measurement of NK Cell Granzyme Secretion.

Granzyme secreted from NK-92 cells was assayed by measuring hydrolysisof Nα-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) essentially asdescribed previously (Takayama H, Trenn G, Sitkovsky M V. A novelcytotoxic T lymphocyte activation assay. J Immunol Methods 1987;104:183-190). Briefly, 50 μL of supernatant was transferred to a 96-wellplate, and 150 μL of BLT solution (0.2 mM BLT; EMD Biosciences, Inc.,and 0.22 mM DTNB; Sigma-Aldrich Co.) (mM=millimolar) inphosphate-buffered saline (PBS, pH 7.2) was added to the supernatant.Absorbance at 410 nm was read after incubation for 30 min at roomtemperature. Results were expressed as percentage of total cellularenzyme content, using the amount obtained in the same number of tritonX-100-lysed cells.

Statistical Analysis.

Statistical significance of the differences between various treatmentgroups was assessed with one-way ANOVA. P values of <0.05 were taken assignificant.

Inhibition of MPO Release from Human Neutrophils

It was found that 100 nM PMA (as a stimulator of inflammatory mediatorrelease) increased human neutrophil MPO release by approximatelythreefold versus control level at 30 min in the PMA control reference,the release of MPO increasing to approximately 5-6 fold after 3 hrs. At30 minutes, relative to the control MPO activity as 100% absent PMA andabsent PMA plus MANS, RNS, or test peptide, MPO activity of the PMAcontrol reference was about 275%, PMA plus 50 μM MANS was about 275%,and 100 μM MANS was about 305%. Thus, the MANS peptide had no detectedeffect at 30 min. However, by 1 hr the higher concentration of MANS (100μM) had a significant inhibitory effect (measured at about 260% ofcontrol) or about 25% reduction in MPO release versus the PMA controlreference level (which was measured at about 340% of control). The 50 μMMANS sample measured about 290% of control or about 15% reductionrelative to the PMA control reference. By 2 hrs and persisting at 3 hrs,the MANS peptide significantly attenuated MPO activity in aconcentration-dependent manner. At 2 hours, the PMA control referenceMPO activity was about 540% of control, the 50 μM MANS (measuring about375% of control) caused an approximately 30% reduction of MPO releaseversus the PMA control reference; and 100 μM MANS (measuring about 295%of control) caused an approximately 45% reduction of MPO release versusthe PMA control reference. At 3 hours, the PMA control reference MPOactivity was about 560% of control, 50 μM MANS (measuring about 375% ofcontrol) caused an approximately 33% reduction of MPO release versus thePMA control reference; 100 μM MANS (measuring about 320% of control)caused an approximately 40% reduction of MPO release versus the PMAcontrol reference. The RNS peptide did not affect PMA-induced MPOrelease at any of the time points or concentrations tested. The datapresented in the table below represents 100 μM concentration of testpeptides and a two hour incubation with 100 nM PMA.

Inhibition of EPO Release from HL-60 Cells

EPO activity in the supernatant of HL-60 clone 15 cells wassignificantly enhanced at 1 and 2 hrs after PMA stimulation. At 1 hour,relative to EPO activity of the control as 100%, the PMA controlreference measured at about 110%; the sample containing 10 μM MANSmeasured at about 95% to give about 15% reduction in EPO activityrelative to the PMA control reference; the sample containing 50 μM MANSmeasured at about 78% to give about 30% reduction in EPO activityrelative to the PMA control reference; and the sample containing 100 μMMANS measured at about 65% to give about 40% reduction in EPO activityrelative to the PMA control reference. At 2 hour, relative to EPOactivity of the control as 100%, the PMA control reference measured atabout 145%; the sample containing 10 μM MANS measured at about 130% togive about 10% reduction in EPO activity relative to the PMA controlreference; the sample containing 50 μM MANS measured at about 70% togive about 50% reduction in EPO activity relative to the PMA controlreference; and the sample containing 100 μM MANS measured at about 72%to give about 50% reduction in EPO activity relative to the PMA controlreference. Thus, at both 1 and 2 hrs, MANS at 50 or 100 μM significantlyattenuated EPO release. The RNS peptide did not affect PMA-enhanced EPOrelease at any of the time points or concentrations tested. The datapresented in the table below represents 50 μM concentration of testpeptides and a two hour incubation with 100 nM PMA.

Inhibition of Lysozyme Release from U937 Cells

Lysozyme secretion by U937 cells was increased by PMA stimulation by 1hr after incubation, and increased even more at 2 hrs. At 1 hour,relative to lysozyme secretion by U937 cells of the control as 100%, thePMA control reference measured at about 210%; the sample containing 10μM MANS measured at about 170% to give about 20% reduction in lysozymesecretion by U937 cells relative to the PMA control reference; thesample containing 50 μM MANS measured at about 170% to give about 20%reduction in lysozyme secretion by U937 cells relative to the PMAcontrol reference; and the sample containing 100 μM MANS measured atabout 115% to give about 45% reduction in lysozyme secretion by U937cells relative to the PMA control reference. At 2 hour, relative tolysozyme secretion by U937 cells of the control as 100%, the PMA controlreference measured at about 240%; the sample containing 10 μM MANSmeasured at about 195% to give about 20% reduction in lysozyme secretionby U937 cells relative to the PMA control reference; the samplecontaining 50 μM MANS measured at about 185% to give about 25% reductionin lysozyme secretion by U937 cells relative to the PMA controlreference; and the sample containing 100 μM MANS measured at about 140%to give about 40% reduction in lysozyme secretion by U937 cells relativeto the PMA control reference. Thus, lysozyme secretion was significantlyattenuated at both 1 and 2 hours post-stimulation by 100 μM of MANS butnot as much by 50 or 10 μM of MANS. The RNS peptide did not affectPMA-enhanced lysozyme secretion at any of the time points orconcentrations tested. The data presented in the table below represents50 μM concentration of test peptides and a two hour incubation with 100nM PMA.

Inhibition of Granzyme Release from NK-92 Cells

The lymphocyte natural killer cell line NK-92 was used to assess releaseof granzyme (Gong J H, Maki G, Klingemann H G. Characterization of ahuman cell line (NK-92) with phenotypical and functional characteristicsof activated natural killer cells. Leukemia 8:652-658, 1994; Maki G,Klingemann H G, Martinson J A, Tam Y K. Factors regulating the cytotoxicactivity of the human natural killer cell line, NK-92. J. Hematother.Stem Cell Res., 10:369-383, 2001; Takayama H, Trenn G, Sitkovsky M V. Anovel cytotoxic T lymphocyte activation assay. J. Immunol. Methods104:183-190, 1987).

Measurement of N K cell granzyme secretion: Granzyme secreted from NK-92cells was assayed by measuring hydrolysis ofNα-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT, EMD Bioscience,Inc.) essentially as described previously (Takayama H, Trenn G,Sitkovsky M V. A novel cytotoxic T lymphocyte activation assay. J.Immunol. Methods 104:183-190, 1987). An aliquot of 50 μL of supernatantwas transferred to a 96-well plate, and 150 μL of 0.2 mM solution of BLTand 0.22 mM DTNB (Sigma-Aldrich Co.) in phosphate-buffered saline (PBS,pH 7.2) was added to the supernatant. Absorbance at 410 nm was measuredafter incubation for 30 min at room temperature. Results were expressedas percentage of total cellular enzyme content, using the amountobtained in the same number of triton X-100-lysed cells.

Because standard granzyme from NK-92 cells was not available to use forquantification, we measured both released and intracellular (from lysedcells) levels of granzyme, and expressed the released granzyme as apercentage of total (intracellular and released) for each. To measureintracellular granzyme from NK-92 cells, appropriate aliquots of 0.1%triton X-100-lysed cells were taken for quantification of the enzyme asdescribed above. All data are expressed as percentage of control tominimize variability between cultures. The data presented in the tablebelow represents 50 μM concentration of test peptides and a two hourincubation with 100 nM PMA.

Cytotoxicity

Because standard None of the treatments generated a toxic response inthe cells, as assessed by LDH retention/release (data not shown) (seealso Park J-A, He F, Martin L D, Li Y, Adler K B. Human neutrophilelastase induces hypersecretion of mucin from human bronchial epithelialcells in vitro via a PKCδ-mediated mechanism. Am J Pathol 2005;167:651-661).

In preliminary experiments, the following peptides which are presentedin the table below demonstrate respective percent inhibition of releaseof MPO from human neutrophils, of EPO from HL-60 clone 15 cells, oflysozyme from U937 cells, and of granzyme from NK-92 cells, whereinMA—signifies the presence of a myristoyl substituent group at thealpha-N-terminal position of the peptide; Ac—signifies the presence ofan acetyl substituent group at the alpha-N-terminal position of thepeptide; H signifies no group attached to the peptide; and NH2 signifiesthe presence of an amide at the C-terminal position. Inhibition data areaveraged from multiple experiments. Qualitative solubility of thepeptides in 0.5 N saline at pH 6.5 is given in mg/mL in Table 3 below.Changing the N-terminal chemical moiety from a myristoyl group can leadto changes in solubility of the peptides disclosed herein in aqueousmedia. For example, changing the myristoyl group to an acetyl groupresults in the increased aqueous solublility shown in Table 3.

TABLE 3Results of Enzyme inhibition assays solubilities for representativepeptides and substituted peptides SEQ % Inhibition mg/mL ID Lyso- Gran-Solu- NO.: N-¹ Amino Acid Sequence C-² EPO zyme MPO zyme bility³ 219 AcAKGE 87.6 7.2 >200  45 Ac AKGEAAAERPGEAAVA 72.3 34.3  37 AcGAQFSKTAAKGEAAAE 56.6 8.4 239 Ac GAQFSKTAAAGE 55.8 37.2  >50 248 AcGAQFSKTAAA 55.2 28.3 >100  91 Ac AAAERPGEAAVA 51.2 29.5  11 AcGAQFSKTAAKGEAAAERPGE 48.8 0.0  79 Ac GAQFSKTAAKGE 46.7 43.3 >100 153 AcRPGEAAVA 45.8 0.0 219 Ac AKGE NH2 45.6 26.8 >200  93 Ac AQFSKTAAKGE NH242.8 51.8  >90 141 Ac SKTAAKGE NH2 42.2 0 >200 241 Ac GAQFSKTAAKGA 40.924.1  >50 143 Ac TAAKGEAA 40.4 0.5 >230 251 Ac AAGE 39.1 36.9 >200 106Ac GAQFSKTAAK 35.7 41.2 25.3 >100 249 Ac GAQFSATAAA 35.7 3.2  <10   1 AcGAQFSKTAAKGEAAAERPGE 33.7 39.8 >250 AAVA 121 Ac GAQFSKTAA 33.3 28.9  >20106 Ac GAQFSKTAAK (all d) 26.9 8.9 40.0 >100 124 Ac FSKTAAKGE NH2 25.356.7 >100  79 Ac GAQFSKTAAKGE NH2 24.7 38.6 26.5  >60 108 Ac QFSKTAAKGENH2 15.7 60.7 >150 179 Ac AAKGEA 10.6 9.2 >150 159 Ac KTAAKGE NH2 024.3 >200 137 Ac GAQFSKTA 0 0 >200  79 H GAQFSKTAAKGE 27.9  >60   1 MAGAQFSKTAAKGEAAAERPGE 46.1 40.8 31.2 76.0    <5.0 AAVA 106 MA GAQFSKTAAK37.4 56.6  >10  11 MA GAQFSKTAAKGEAAAERPGE 33.6 99 179 MA AAKGEA 31.428.6    <1.0  37 MA GAQFSKTAAKGEAAAE 30.3 99    >2.0  79 MA GAQFSKTAAKGE25.2 85.2 43.2    >2.0  91 MA AAAERPGEAAVA 21.6 98  <20  45 MAAKGEAAAERPGEAAVA 18.1 98  >80 153 MA RPGEAAVA 0 99  15 MASKTAAKGEAAAERPGEAAVA 0 99  >80 143 MA TAAKGEAA 0 80.2    <1.0 219 MAAKGE 0 28.6    <1.0 232 MA GTAPAAEGAGAEVKRASAEA 0 0 0 29.5  >15 KQAF 234MA GAQFSKTKAKGE 65.2    >3.0 ¹N- =N-terminal group ²C- =C-terminal group³0.5 N Saline, pH 6.5

Example 2 In Vivo Inhibition of Lipopolysaccharide (LPS)-Induced LungInflammation by MANS and Related Peptides

This example was performed essentially according to methods described byCox, G, Crossley, J., and Xing, Z.; Macrophage engulfment of apoptoticneutrophils contributes to the resolution of acute pulmonaryinflammation in vivo; Am. J. Respir. Cell Mol. Biol. 12:232-237, 1995;Hirano S., Quantitative time-course profiles of bronchoalveolar lavagecells following intratracheal instillation of lipopolysaccharide inmice, Ind. Health 35:353-358, 1997; and Ulich T R, Watson L R, Yin S M,Guo K Z, Wang P, Thang H, and del Castillo, J. Am. J. Pathol.138:1485-1496, 1991.

Thus, six to seven week old CD1 female mice weighing 15-20 grams wereobtained from Charles River laboratories and housed in groups of 5 miceper cage. The animals received standard rodent diet and filtered waterad libitum. The animals were housed under NIH prescribed guidelines atstandard temperature (64° to 79° F.) and relative humidity of 30 to 70%.

Five treatment groups of mice, with 5 animals in each group, weretreated either with PBS followed by PBS, with PBS followed by LPS, with(myristoylated) MANS peptide followed by LPS, with acetylated peptide ofSEQ ID NO: 1, followed by LPS, or with acetylated peptide of SEQ ID NO:106, followed by LPS.

Intranasal peptide instillation pre-treatment: A peptide of theinvention to be evaluated in vivo for its ability to inhibit or reduceLPS-induced lung inflammation was dissolved in PBS at a concentration of1 mM. Animals, anesthetized with 0.8% isofluorane by inhalation, werepretreated with 2×10 μL intranasal bolus of the peptide solution intoone nostril 30 minutes prior to subsequent instillation with LPS.

Intranasal LPS instillation: Lipopolysaccharide (LPS) Endotoxin(Escherichia coli Serotype 011:B4 derived endotoxin; Sigma, St Louis,Mo.; see Sigma product information sheet L4130 titledLipopolysaccharides from Escherichia coli 011:B4) was dissolved intophosphate buffered saline (PBS) at 2,500 μg/mL. To expose animals toendotoxin, a 10 μL intranasal bolus of 2,500 μg/ml endotoxin solutionwas administered to animals which had been anesthetized with 0.8%isofluorane by inhalation. The 10 μL bolus was applied into one nostril.Animals were monitored for labored breathing, lethargy, and decreasedwater/food intake following the endotoxin instillations.

Bronchoalveolar Lavage (BAL): Six hours after the last instillation, theanimals were anesthetized (90 mg/kg Nembutal) and sacrificed byexsanguination. The lung was serially lavaged 2 times with 1.0 mLaliquots of PBS. The collected BAL fluid was centrifuged to remove thecells for subsequent counting and differential analysis. Recoveredlavage fluid was used for analysis of total protein, myeloperoxidase(MPO), LDH, and hemoglobin.

Analysis: Aliquots of the BAL fluid were used immediately to assay forthe levels of LDH, total protein, or hemoglobin using the COBAS Faraanalyzer (COBAS FARA II automated analyzer; Roche Diagnostic SystemsInc., Montclair, N.J.). An aliquot of BAL fluid was frozen at −80° C.for subsequent quantitation of myeloperoxidase (MPO) with amouse-specific ELISA assay (Cell Sciences, Inc., Canton, Mass.). BALdata were analyzed by standard techniques to examine differences betweenthe control and treatment groups. Results demonstrating inhibition orreduction of inflammation by Test peptide are provided in the followingtables.

TABLE 4Average values of markers of inflammation in the presense of MANS peptide,MA-GAQFSKTAAKGEAAAERPGEAAVA, SEQ ID NO.: 1 Total Total % Neutro- TotalTreatment cells neutrophils phils of MPO Protein LDH Hb Regime countedcounted total cells (ng/mL) (ug/ml) (units/L) (g/dl) PBS/PBS 157,020  29317 18.7  3.28 125.60 68.20 0.00 n = 5 PBS/LPS 264,200 110,061 41.728.98 272.40 60.40 0.19 n = 5 MANS/LPS 208,457  64,481 30.9  9.49 175.0068.57 0.05 n = 7

TABLE 5Average values of markers of inflammation in the presence of an N-terminalacetylated analog of MANS peptide, Ac-GAQFSKTAAKGEAAAERPGEAAVA, SEQ ID NO: 1Total % Neutro- Total Treatment Total Cell Neutrophil phils of MPOProtein LDH Hb Regime Counts counts total counts (ng/mL) (μg/mL) (units/L) (g/dL) PBS/PBS  89,440  19,770 22.1  5.45 230.6  84.0 0.00 n =5 PBS/LPS 251,360 164,578 65.5 37.90 153.4  89.9 0.01 n = 5 Ac-SEQ ID254,400 105,499  41.47 30.79 182.75 74.5 0.01 NO.: 1/LPS n = 5

TABLE 6Average values of markers of inflammation in the presence of acetylated peptideAc-GAQFSKTAAK, SEQ ID NO.: 106 Total % Neutro- Total TreatmentTotal Cell Neutrophil phils of MPO Protein LDH Hb Regime Counts countstotal counts (ng/mL) (μg/mL)  (units/L) (g/dL) PBS/PBS 312,620 66,52121.3 4.88 113.8  61.80 0.00 n = 5 PBS/LPS 327,680 80,077 24.4 7.19 116.4 78.20 0.00 n = 5 Ac-SEQ ID 305,688  9,170  3.0 1.50 131.0 106.86 0.00NO.: 106/ LPS n = 5

TABLE 7 Inhibition of markers of inflammation by MANSpeptide (Myr-SEQID NO:1), test peptides (Ac-GAQFSKTAAKGEAAAERPGEAAVA), SEQ ID NO: 1, andAc-GAQFSKTAAK, SEQ ID NO: 106, relative to PBS/LPS treatment:Inhibition of Treatment neutrophil Inhibition of Regime migration MPOMANS/LPS 41.4% 67.2%  SEQ ID NO: 1/LPS 35.9% 18.75% Ac-SEQ ID NO.  88.5%79.1%  106/LPS

PBS/PBS indicates only PBS control was administered, and no LPSendotoxin was added to stimulate chemotactic neutrophil migration;PBS/LPS indicates LPS (endotoxin) was added to stimulate chemotacticneutrophil migration; MANS/LPS indicates pretreatment with MANS peptidein PBS followed by LPS stimulation to induce neutrophil migration. Thepercent of neutrophils in the total cell count in the LPS treatmentgroups was reduced from 41.7% to 30.9% by treatment with MANS peptide;from 65.5% to 41.47% by treatment with the peptideAc-GAQFSKTAAKGEAAAERPGEAAVA, SEQ ID NO. 1; from 24.4% to 3.0% bytreatment with the peptide Ac-GAQFSKTAAK, SEQ ID NO. 106. The measuredMPO levels in the LPS treatment groups was reduced from 28.98 ng/mL to9.49 ng/mL by treatment with MANS peptide; from 37.9 ng/mL to 30.79ng/mL by treatment with the peptide with acetylated SEQ ID NO:1 and from7.19 ng/mL to 1.50 ng/mL by treatment with the peptide with acetylatedSEQ ID NO:106.

Example 3 Mouse Model of Ozone-Induced COPD

Oxidative stress by chemical irritants such as ozone is a widelyrecognized feature of chronic obstructive respiratory disease (COPD).See: Repine J E, Bast A, Lankhorst I, and the Oxidative Stress StudyGroup, Am. J. Respir. Crit. Care Med. 156:341-357, 1997; and alsoHarkema J R and Hotchkiss J A, Toxicology Letters, 68:251-263, 1993.

Ten-week-old Balb/C female mice were obtained from Charles Riverlaboratories and housed under NIH guidelines in groups of 5 per cage.The animals received standard rodent diet and filtered water ad libitum.Three treatment groups of mice, 5 animals in each group, were eachanesthetized by intraperitoneal injection of Ketamine (100 mg/kg) andXylazine (20 mg/kg) and then pretreated by intratracheal administrationwith 25 μL of either PBS alone, or a solution of 1.0 mM MANS peptide inPBS, or a solution of a 1.0 mM of an acetylated MANS-fragment-peptideAc-GAQFSKTAAK designated as acetylated SEQ ID NO: 106 in PBS. After 30minutes, the animals were then placed in the appropriate custom-madechamber for ozone or forced air exposures. The animals were exposed toozone for 2 hours (at ozone concentrations of 1-10 ppm by a slightlymodified method described by Haddad et al, 1995. (Haddad E-B, Salmon M,Sun J, Liu S, Das A, Adcock I, Barnes P J, and Chung K F, FEBS Letters,363:285-288, 1995). The ozone was generated using an ozone generatorapparatus model OL80F/B from OzoneLab, Burton, British Columbia, Canada.Ozone concentration was continuously monitored using a TeledynePhotometric O3 Analyzer (model 400E, Teledyne Instruments, City ofIndustry, Calif.). Two additional groups of mice, each without anypretreatment, were either exposed to ozone under the same conditions orexposed to forced air under conditions similar to the ozone treatmentgroup but absent ozone. After exposure, the animals were sacrificed byexsanguination and the lungs were serially lavaged 2 times with 1.0 mLaliquots of PBS. The collected bronchoalveolar lavage (BAL) fluid wascentrifuged to remove the cells for subsequent counting and differentialanalysis. Recovered lavage fluid was used for protein and additionalanalysis of IL-6, IFNγ, and KC (murine IL-8 analog) by ELISA assay(assay kits obtained from R&D Systems, Minneapolis, Minn.).

The percent inhibition of neutrophil migration into the BAL fluid as afunction of treatment groups and relative to a control group treatedwith PBS alone are provided in the table.

TABLE 8 Inhibition of ozone-induced neutrophil migrationby MANS peptide and by peptide acetylated SEQ ID NO: 106, Ac-GAQFSKTAAK.% Inhibition of neutrophil migration into Treatment Group BAL fluidMANS + Ozone 93.0 Ac-SEQ ID NO: 106 + 81.2 Ozone PBS + OzoneNot applicable Forced air alone Not applicable

Concentrations of IL-6 in pg/mL in BAL fluid, as a function ofintratracheal injection pretreatment and subsequent treatment withozone, were obtained as follows. IL-6 levels were found to be:approximately 364.5 pg/mL in a group of mice pretreated with MANSpeptide and then exposed to ozone; approximately 130.4 pg/mL in a groupof mice pretreated with acetylated MANS-fragment-peptide, Ac-GAQFSKTAAK(SEQ ID NO: 106), and then exposed to ozone; approximately 1041.3 pg/mLin a group of mice pretreated with PBS and exposed to ozone;approximately 43.2 pg/mL in a group of mice exposed directly to forcedair without any pretreatment.

Concentrations of KC in pg/mL in BAL fluid, as a function ofintratracheal injection pretreatment and subsequent treatment withozone, were obtained as follows. KC levels were found to be:approximately 183.6 pg/mL in a group of mice pretreated with MANSpeptide and then exposed to ozone; approximately 159.7 pg/mL in a groupof mice pretreated with acetylated MANS-fragment-peptide, Ac-GAQFSKTAAK(SEQ ID NO:106), and then exposed to ozone; approximately 466.6 pg/mL ina group of mice pretreated with PBS and exposed to ozone; approximately36.3 pg/ml in a group of mice exposed to forced air withoutpretreatment.

Concentrations of IFNγ in pg/mL in BAL fluid as a function ofintratracheal injection pretreatment and subsequent treatment with ozonewere obtained as follows. IFNγ levels were found to be: approximately7.4 pg/mL in a group of mice pretreated with MANS peptide and thenexposed to ozone; approximately 3.6 pg/ml in a group of mice pretreatedwith acetylated MANS-fragment-peptide, Ac-GAQFSKTAAK (SEQ ID NO:106),and then exposed to ozone; approximately 8.6 pg/mL in a group of micepretreated with PBS and exposed to ozone; and approximately 5.0 pg/mL ina group of mice exposed to forced air.

Administration of ozone to mice significantly increased infiltratedneutrophil cell numbers, as well as IL-6 and KC levels in the BAL. Incomparison to the control group in which the mice were pretreated withPBS, the group pretreated with MANS peptide and the group pretreatedwith acetylated peptide, Ac-GAQFSKTAAK, acetylated SEQ ID NO:106. eachexhibited reduced neutrophil cell infiltration in the BAL fluid afterozone exposure (e.g., 93%±10% and 81%±10%, respectively vs. PBScontrol). In parallel, MANS peptide and acetylated peptide acetylatedSEQ ID NO:106 also markedly diminished KC concentrations (e.g.,65.8%±10% and 71.3%±10%, respectively, vs. PBS control) and IL-6 levels(e.g., 67.8%±15%, MANS and 91.3%±15% acetylated SEQ ID NO:106 vs. PBScontrol) after ozone exposure but had little effect on interferon-γlevels. Collectively, these data evidence that MANS peptide andacetylated SEQ ID NO:106 peptides markedly diminish or inhibitozone-induced neutrophil migration into the airways as well as decreaseselective chemokine and cytokine. The IL-6 levels in the BAL fluids fromanimals pretreated with MANS peptide or acetylated peptide SEQ ID NO:106showed approximately 68% and 91% inhibition, respectively, compared tothose pretreated with PBS. Also the KC levels in the BAL fluids fromanimals pretreated with MANS peptide or acetylated peptide SEQ ID NO:106showed approximately 65% and 71% inhibition compared to those pretreatedwith PBS.

Example 4 Chronic Bronchitis Model

The procedure is described by Voynow J A, Fischer B M, Malarkey D E,Burch L H, Wong T, Longphre M, Ho S B, Foster W M, Neutrophil Elastaseinduces mucus cell metaplasia in mouse lung, Am. J. Physiol. Lung CellMol. Physiol. 287:L1293-L1302, 2004 and is followed to develop a modelof chronic bronchitis in the mouse. Specifically, goblet cellhyperplasia, a signature pathological feature of chronic bronchitis, isinduced by chronic exposure of mice to human Neutrophil Elastase (NE)instilled into the airways.

Human NE are aspirated intratracheally by male Balb/c mice. A total of30 mice (about 25-30 g in weight) are obtained commercially from asupplier such as Jackson Laboratories, Bar Harbor, Me. The mice aremaintained on a 12 hr diurnal cycle, with food and water provided adlibitum. The animals receive NE by oropharyngeal aspiration on days 1,4, and 7. Immediately after inhalational anesthesia with isofluorane(IsoFlo from Abbott Laboratories and Open-Circuit Gas Anesthesia Systemfrom Stoelting), animals are suspended by their upper incisors on a 60°incline board, and a liquid volume of human NE [50 ug (43.75 units)/40μL PBS (Elastin Products, Owensville, Mo.) is delivered with theanimal's tongue extended to the distal part of the oropharynx. With thetongue extended, the animal is unable to swallow, and the liquid volumeis aspirated in the respiratory tract.

At 7 days after the last NE exposure, when the goblet cell hyperplasiamodeling the airways in chronic bronchitis is at its maximum (see Voynowet al, 2004), mice (5 animals per group) are instilled intra-tracheallywith 50 μL of either PBS (as control), or 100 uM of a solution of MANSpeptide, a solution of RNS peptide, or a solution of a peptide such asacetylated peptide SEQ ID NO:106 dissolved in PBS. Fifteen minuteslater, mucus secretion is triggered by administration of methacholine,using a Buxco system Nebulizer to provide a fine aerosol deliveringmethacholine at approximately 60 mM for 3 min. Fifteen minutes aftermethacholine administration, mice are sacrificed by inhalationalexposure to 100% CO2 gas.

Histochemistry. After exposures described above, lungs from animals areflushed to remove blood, then are inflated with OCT (Optimum CuttingTemperature medium (Sakura Finetck, Torrance, Calif.), half diluted insaline. The lungs are immersed in 10% formaldehyde in PBS overnight at4° C., and processed to paraffin wax. Five μm sections are treated withPeriodic acid Schiff/haematoxylin to stain mucins in the airways, forexample as described by Singer M, Vargaftig B B, Martin L D, Park J J,Gruber A D, Li Y, Adler K B, A MARCKS-related peptide blocks mucushypersecretion in a murine model of asthma., Nature Medicine 10:193-196,2004.

Histological mucus index. A histological mucus index (Whittaker L, NiuN, Temann U-A, Stoddard A, Flavell R A, Ray A, Homer R J, and Cohn L,Interleukin-13 mediates a fundamental pathway for airway epithelialmucus induced by CD4 T cells and interleukin-9, Am. J. Respir. Cell Mol.Biol. 27:593-602, 2002) is performed on AB/PAS-stained sections thatinclude both central and peripheral airways. The slides are examinedwith a 10× objective, and images captured with a digital camera. Aminimum of four representative cross- or sagittally sectioned airways isimaged per animal. Only airways where the complete circumference of theairway can be visualized and included in the image are analyzed. Airwaysthat open directly in an alveolar space are not included. The extent ofPAS-positive staining in each airway imaged will be semi-quantitativelydetermined by an examiner who does not know the treatment conditions foreach section, using the following five-tier grading system: grade 0, noPAS staining; grade 1, 25% or less of the airway epithelium has PASstaining; grade 2, 26-50% of the airway epithelium has PAS staining;grade 3, 51-75% of the airway epithelium has PAS staining; and grade4, >75% of the airway epithelium has PAS staining. This grading systemis used to calculate a mucus index score for each group, and results arepresented as means±SE.

All results are presented as means±standard error (n=5 animals, 10-20sections for each). Significance levels will be calculated using one wayANOVA followed by Scheffe's test, using SPSS 6.1 software(*=significance between data with a threshold of p<0.05).

Example 5 In Vivo Assays

The objective of the following set of experiments is to establish theeffects of the peptides of this invention after in vivo delivery, eitherby local instillation at the site of inflammation or i.v. injection, oninflammation compared to the control peptides such as RNS. Two modelsare useful for this determination: (i) the murine air pouch inflammationmodel and (ii) the murine thioglycollate induced peritonitis model. Bothare well-characterized models of inflammation in which neutrophils havean essential role. The air pouch model enables determination of theeffects of the peptides on a short time course of inflammation(approximately 4 hrs) and the peritonitis model is useful with respectto a longer time course of inflammation (approximately 24 hrs).

Overall Experimental Design:

Four studies, two for each model, one testing i.v. delivery of thepeptides and one testing local delivery of the peptides are useful forstudying the effect of the peptides disclosed in this application. Eachstudy consists of 2 experimental groups, a non-inflamed control (treatedwith vehicle) and an inflamed group (i.e., treated with an inflammatorystimulus). Each group is divided into 5 and optionally 6 treatmentsubgroups, n=6 for each subgroup. Treatments subgroups are, for example:vehicle, MANS, RNS, test peptide, optionally a peptide having ascrambled sequence of the test peptide which scrambled sequence aredubbed “peptide-SCR”, and dexamethasone. Dexamethasone serves as areference anti-inflammatory agent. The selection of appropriate dosesfor i.v. injection or local instillation are determined from preliminarydose response experiments. Tentative doses based on the inhibitoryactivity of MANS in human neutrophils are: 1 mg/kg for i.v. deliveryadministered once or a final concentration of 50 μM delivered locally(into the air pouch or i.p.). The dose for i.v. delivery are chosenassuming a volume of distribution of 2 L/kg.

Air Pouch Inflammation Model:

Assays for neutrophil infiltration and inflammation in the mouse airpouch are performed as described in Clish C B, O'Brien J A, Gronert K,Stahl G L, Petasis N A, Serhan C N. Local and systemic delivery of astable aspirin-triggered lipoxin prevents neutrophil recruitment invivo. Proc Natl Acad Sci USA. 1999 Jul. 6; 96(14):8247-52. Thus, whitemale BALB/c mice (6-8 wk) are anesthetized with isoflurane, and dorsalair pouches are raised by injecting 3 ml of sterile air subcutaneouslyon days 0 and 3. On day 6 and while the mice are anesthetized withisoflurane, vehicle, MANS, RNS, test peptide, or optionally peptide-SCRare delivered as a bolus injection either i.v. into the tail vein in 100μL of sterile 0.9% saline or locally into the air pouch in 900 μL ofPBS−/− (Dulbecco's Phosphate Buffered Saline without magnesium orcalcium ions, BioWhittaker). Dexamethasone (Sigma) delivered i.v. as 0.1mg/kg in 100 μl sterile 0.9% saline or locally as 10 μg in 900 μL ofPBS−/−, serves as a reference anti-inflammatory agent. Inflammation inthe air pouch is induced by local injection of recombinant murine tumornecrosis factor α (TNF-α, 20 ng) (Boehringer Mannheim) dissolved in 100μL of sterile PBS. While the mice are anesthetized with isoflurane, theair pouches are lavaged twice with 3 mL of sterile PBS 4 hr after theinitial TNF-α injection. Aspirates are centrifuged at 2,000 rpm for 15min at 23° C. The supernatants are removed, and the cells suspended in500 μL of PBS. Aliquots of the supernatant are assayed for inflammatorymediator concentrations (optionally except for TNFα), MPO activity, andlipid peroxidation.

Total leukocytes are enumerated in the cell suspension by lightmicroscopy using a hemocytometer. Resuspended aspirate cells (50 μL) areadded to 150 μL of 30% BSA and centrifuged onto microscope slides at2,200 rpm for 4 min by using a cytofuge. Differential leukocyte countsare determined in cytospins stained with Wright Giemsa stain and used tocalculate the absolute number of each leukocyte per air pouch exudate.For microscopic analysis, tissues are obtained with a 6-mm tissue biopsypunch (Acu-Punch, Acuderm) and fixed in 10% buffered formaldehyde.Samples are then embedded in paraffin, sliced and stained withhematoxylin-eosin. Neutrophils are enumerated in histological sectionsby counting number of cells/hpf. Distant dermis serve as a control forthe inflamed air pouch dermis.

Data are presented as total number of neutrophils, monocytes,eosinophils, basophils, and lymphocytes per exudate or the numberneutrophils per tissue high power field. Values are reported as themean±SEM (n=6). The significance of any treatment on migration aredetermined by ANOVA. P<0.05 is to be considered significant.

Example 6 Inflamed Peritoneum Model

Male BALB/c mice (6-8 wk) are used and the thioglycollate-inducedperitonitis models performed as described in Tedder T F, Steeber D A,Pizcueta P. L-selectin-deficient mice have impaired leukocyterecruitment into inflammatory sites. J Exp Med. 1995 Jun. 1;181(6):2259-64. Vehicle, MANS, RNS, test peptide, and optionallypeptide-SCR are delivered as a bolus injection either into the tail veinin 100 μL of sterile 0.9% saline or locally into the peritoneum 900 μlof PBS−/− immediately prior to i.p. injection of thioglycollate.Dexamethasone delivered i.v. as 0.1 mg/kg in 100 μL sterile 0.9% salineor locally as 10 μg in 900 μl of PBS−/−, serves as a referenceanti-inflammatory agent. Inflammation is induced by injection of 1 mL ofthioglycollate solution (3% wt/vol; Sigma Immunochemicals)intraperitoneally into the mice. Mice are humanely euthanized 24 hrsfollowing induction of inflammation and 5 mL of warm (37° C.˜medium(RPMI 1640, 2% FCS, and 2 mM EDTA) injected into the peritoneum followedby gentle massage of the abdomen. Aspirates of the abdominal lavagefluid are centrifuged at 2,000 rpm for 15 min at 23° C. The supernatantsare removed, and the cells suspended in 500 μL of PBS. Aliquots of thesupernatant are assayed for MPO activity, inflammatory mediatorconcentrations, and lipid peroxidation.

Total leukocytes are enumerated in the cell suspension by lightmicroscopy using a hemocytometer. Resuspended aspirate cells (50 μL) areadded to 150 μL of 30% BSA and centrifuged onto microscope slides at2,200 rpm for 4 min by using a cytofuge. Differential leukocyte countsare determined in cytospins stained with Wright Giemsa stain and used tocalculate the absolute number of each leukocyte per air pouch aspirate.

Data are presented as total number of neutrophils, monocytes,eosinophils, basophils, and lymphocytes per exudate. Values are reportedas the mean±SEM (n=6). The significance of any treatment on migration isdetermined by ANOVA. P<0.05 is to be considered significant.

Degranulation:

Myeloperoxidase is used as a marker of degranulation. Myeloperoxidaseactivity in the cell supernatant obtained from the air pouch orperitoneal lavage fluid is assayed and analyzed as described above usingthe TMB method.

Inflammatory Mediator Concentrations:

Concentrations of the key pro-inflammatory mediators TNFα, IL-1β, IL-10,IL-6, KC, and PGE2 in air pouch and peritoneal lavage fluid aredetermined using commercial ELISA kits (R&D Systems) according to themanufactures instructions.

Lipid Peroxidation:

The concentration of F2-isoprostanes is a sensitive and specific measureof oxidative injury resulting from release of reactive oxygenintermediates from neutrophils and other cells {Milne G L, Musiek E S,Morrow J D. F2-isoprostanes as markers of oxidative stress in vivo: anoverview. Biomarkers. 2005 November; 10 Suppl 1:S10-23}. F2-isoprostaneconcentration is determined in air pouch and peritoneal exudatesupernatants using a commercially available ELISA (8-Isoprostane EIA,Cayman Chemical) according to the manufactures instructions.

End Point:

The experiment is considered to be successful if either local orsystemic delivery of the test peptide reduces inflammation by one ormore of the above measures of inhibition of release of inflammatorymediator.

The active fragment peptides of this invention inhibit neutrophil influxinto and degranulation in inflamed air pouch or peritoneum, resulting inreduced MPO activity, reduced lipid peroxidation, and reducedinflammatory mediator production.

The foregoing examples are illustrative of the present invention and arenot to be construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

1.-57. (canceled)
 58. A method for treating or preventing adultrespiratory distress syndrome (ARDS), acute pulmonary inflammation, oracute thermal injury in a subject in need thereof, the method comprisingadministering to the subject an effective amount of a peptide comprisingan amino acid sequence selected from the group consisting of: SEQ IDNOs: 106, 1, 11, 37, 45, 79, 91, 93, 108, 121, 124, 141, 143, 153, 219,234, 239, 248, 241, and
 251. 59. The method of claim 58, wherein theN-terminal amino acid of the peptide is chemically modified byacylation.
 60. The method of claim 58, wherein the N-terminal amino acidof the peptide is chemically modified by myristoylation or acetylation.61. The method of claim 58, wherein the C-terminal amino acid of thepeptide is chemically modified by amidation.
 62. The method of claim 58,wherein the peptide consists of acetyl-SEQ ID NO: 106 or acetyl-SEQ IDNO:
 121. 63. The method of claim 58, wherein the peptide is administeredby inhalation, parenteral administration, pulmonary administration,nasal administration, or oral administration.
 64. The method of claim58, wherein the peptide is administered in the form of an aerosol. 65.The method of claim 58, wherein the peptide is administered in the formof a dry powder.
 66. The method of claim 58, wherein the peptide isadministered by a dry powder inhaler, metered dose inhaler, ornebulizer.
 67. The method of claim 58, wherein the subject is a human.68. The method of claim 58, further comprising administration to saidsubject of a second therapeutic agent selected from the group consistingof an antibiotic, an antiviral compound, an antiparasitic compound, ananti-inflammatory compound, and an immunomodulator.